Journal: Life Science Alliance

Article Title: Targeted recruitment of USP15 enhances CTLA4 surface levels and restricts its degradation

doi: 10.26508/lsa.202503563

Figure Lengend Snippet: (A) Schematic illustration of the RapTag system. The A/C heterodimeriser is used to induce artificial proximity between a protein of interest (POI) fused to the FRB-T2098L (FRB*) and a DUB tagged with FKBP12 (FKBP). (B) Schematics of WT and catalytically inactive (C>S mutation; CS) DUB and POI constructs used in the RapTag system. Restriction sites (RE) allow swapping of the DUB and POI modules for target validation and DUB/POI pairing; FLAG- and HA-tags are used for easy detection. (C) Cartoon of isogenic cell lines generated for doxycycline (Dox)-inducible, equal expression of WT and mutant DUBs. (D) Schematic of the RapTag experimental protocol. DUB expression is induced with Dox and the POI is transfected into the cells. After 18 h, the medium is exchanged and the A/C dimeriser (no Dox) is added for another 24 h before harvesting for a variety of readouts (Western blotting, Immunofluorescence, HiBiT assay). FRB, FKBP-rapamycin binding domain (FRB) of the mammalian target of rapamycin (mTOR) kinase; FKBP12, rapamycin binding protein FK506-binding protein; FRT, Flp recombination target.

Article Snippet: Antibodies and other reagents used were as follows: anti-HA (MMS-101P; WB 1:1,000; Covance), anti-HA (NB600-362; WB 1:10,000; IF 1:250; Novus Biologicals), anti-HA (3724; WB 1:1,000; Cell Signaling Technology), anti-Actin (66009; WB 1:5,000; Proteintech), anti-HiBiT (N720A; WB 1:1,000; IF 1:1,000; Promega), anti-GAPDH (2118S; WB 1:1,000; Cell Signaling Technologies), anti-USP15 (A300-923A; WB 1:1,000; Bethyl Laboratories), anti-FLAG (F3165 or F1804; WB 1:1,000; IF 1:1,000; M2 Sigma-Aldrich), anti-DYKDDDDK (FLAG) Tag (D6W5B) (14793; IF 1:1,000; Cell Signaling Technology), anti-ubiquitin FK2 (PW8810; WB 1:500; Enzo), anti-USP8 antibody (AF7735; WB 1:500; R&D Systems), Wheat Germ Agglutinin (WGA) Alexa Fluor 647 (gift from Shankar Varadarajan, Liverpool), A/C Heterodimeriser (635055, 500 nM unless otherwise indicated; Takara/Clontech), doxycycline (D9891, 0.1 μg/ml; Sigma-Aldrich), cycloheximide (C7698, 100 μg/ml; Sigma-Aldrich), concanamycin A (C9705, 100 nM; Sigma-Aldrich), epoxomicin (324800, 1 μM; Merck Millipore), TAK-243 (S8341, 1 μM; Selleckchem), and Poly-L-lysine (P4832, solution, 0.01%; Merck Millipore).

Techniques: Mutagenesis, Construct, Biomarker Discovery, Generated, Expressing, Transfection, Western Blot, Immunofluorescence, Binding Assay

Journal: Science Advances

Article Title: Immunosuppressive macrophages determine the effect of cellular senescence on tumor progression

doi: 10.1126/sciadv.adx2988

Figure Lengend Snippet: ( A ) Schematic representation of the construct used to generate SuSe mice. The bicistronic construct consists of a fusion protein FRB–caspase 8 (Casp8), followed by an internal ribosome entry site (IRES) and the mCherry autofluorescent protein, all under the control of the 1200–base pair (bp)–long mouse p16 promoter. Similarly, the bicistronic construct, FKBP–caspase 8, IRES, and GFP, is expressed under the control of the 1400-bp-long IL-6 promoter. The presence of mCherry and GFP allows for the identification of cells expressing high levels of p16 and IL-6, respectively. The addition of AP21967 induces the heterodimerization of FRB and FKBP, resulting in caspase 8–mediated apoptosis. ( B ) SuSe mice were crossed with MMTV-PyMT mice to characterize the effect of senescence during breast cancer progression. ( C ) SuSe/PyMT mice were euthanized at different time points corresponding to the indicated tumor stages. Samples from mammary glands were processed and stained for SA-β-Gal or analyzed by immunofluorescence to visualize the expression of the reporters under the control of p16 and/or IL-6 promoters, mCherry, and GFP, respectively. Results are expressed as averages ± SDs of three mice. ns, not significant. ( D ) The same mice were treated with vehicle or AP21967 every 3 days, starting at 4 weeks. At 6 weeks, mice were euthanized and analyzed as in (C).

Article Snippet: PyMT/SuSe mice were injected intraperitoneally (ip) every 3 days with 0.2 μg/g of body weight of AP21967 (#635055, Takara) from the fourth or ninth week of life until week 13.

Techniques: Construct, Control, Expressing, Staining, Immunofluorescence

Journal: Science Advances

Article Title: Immunosuppressive macrophages determine the effect of cellular senescence on tumor progression

doi: 10.1126/sciadv.adx2988

Figure Lengend Snippet: ( A ) SuSe/PyMT mice were treated with vehicle or AP21967 either at 4 weeks (early senolysis) or at 9 weeks (late senolysis), and tumor volumes were monitored at the specified time points. Typically, each mouse developed four or five tumors, and we calculated the average tumor volume per mouse. The results shown are the means of these average tumor volumes ± SD. Two-way analysis of variance (ANOVA) was used to determine statistical significance. ( B ) At the end of the experiment, mice were euthanized, and lungs were fixed, embedded in paraffin, and sectioned as indicated. ( C ) Representative examples of staining with anti–pan-keratin to visualize metastases. ( D ) The numbers of metastases (Mets) were determined in every slide and are represented as averages ± SD. t test was used to determine statistical significance. ( E ) The areas of all metastases are represented. Average number of metastases is represented in the y axes, and average metastasis areas are represented as circle areas. t test was used to determine statistical significance.

Article Snippet: PyMT/SuSe mice were injected intraperitoneally (ip) every 3 days with 0.2 μg/g of body weight of AP21967 (#635055, Takara) from the fourth or ninth week of life until week 13.

Techniques: Staining

Journal: Science Advances

Article Title: Immunosuppressive macrophages determine the effect of cellular senescence on tumor progression

doi: 10.1126/sciadv.adx2988

Figure Lengend Snippet: ( A ) Uniform Manifold Approximation and Projection (UMAP) of single cells from tumors treated as shown in , showing distinct clusters predominantly determined by cell type and status. T reg , regulatory T cells. ( B ) UMAP plots at 4 and 9 weeks for vehicle- and AP21967-treated mice, illustrating the distribution of different cell populations. ( C ) Total number of cells in each population that increased (Up) or decreased (Down) between 4 and 9 weeks and with or without AP21967 treatment. ( D ) Quantification of the log 2 fold change (FC) in the number of each cell type at 9 weeks after AP21967 treatment compared to vehicle treatment. ( E ) Immunohistochemical analyses of the infiltration of endothelial cells (CD31 + ) and lymphocytes (CD8 + ) in tumors from control mice and tumors from mice treated with AP21967, with accompanying bar graphs showing the percentage of CD31 + and CD8 + cells. t test was used to determine statistical significance.

Article Snippet: PyMT/SuSe mice were injected intraperitoneally (ip) every 3 days with 0.2 μg/g of body weight of AP21967 (#635055, Takara) from the fourth or ninth week of life until week 13.

Techniques: Immunohistochemical staining, Control

Journal: Science Advances

Article Title: Immunosuppressive macrophages determine the effect of cellular senescence on tumor progression

doi: 10.1126/sciadv.adx2988

Figure Lengend Snippet: ( A ) Tumor samples from mice treated with vehicle or AP21967 at 9 weeks were homogenized, and the levels of the indicated cytokines were analyzed using a cytokine array. The log 2 FC (AP21967/vehicle) for each cytokine is presented. ENA-78, epithelial-derived neutrophil-activating peptide 78; GROα, growth-regulated oncogene-alpha. ( B ) Levels of CCL2 and CXCL1 were quantified by ELISA from the same tumor homogenates as in (A). ( C ) UMAP plots showing the expression of the most up-regulated cytokines (CCL2, CCL3, and CCL7) and their receptors (CCR1, CCR2, and CCR5) in different cell populations. Relevant cell clusters are indicated. N, neutrophils; MØ, macrophages; E, endothelial cells; F, fibroblasts. ( D ) Flow cytometry analysis of myeloid cells purified from tumors obtained from SuSe mice treated with vehicle or AP21967. We identified TAMs and cells positive for the myeloid marker CD11B and for the macrophage marker F4/80. ( E ) The same cells as in (D) were cultured for 24 hours—in the presence of vehicle or anti-CCL2—and the presence of CCL2 in the conditioned medium was determined by ELISA. ( F ) Schematic representation of the specificities of receptors for CCL2, CCL3, and CCL7. ( G ) Flow cytometry analysis of CCR2 in the same cells as in (D). FSH-H, forward scatter height.

Article Snippet: PyMT/SuSe mice were injected intraperitoneally (ip) every 3 days with 0.2 μg/g of body weight of AP21967 (#635055, Takara) from the fourth or ninth week of life until week 13.

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Purification, Marker, Cell Culture

Journal: bioRxiv

Article Title: Blockade of associative spine enlargement impairs awake cortical function

doi: 10.1101/2025.11.06.686434

Figure Lengend Snippet: a , Distributions of detected GCaMP7f event amplitudes (ΔF/F₀) per minute before (left) and after (center) A/C heterodimerizer administration, and event frequency (right). n = 493 cells (5 mice). Mean amplitudes were equivalent. Wilcoxon signed-rank test: P = 0.29 (W = 4.8 × 10⁴). b , Spine Ca²⁺ transients (red) isolated from adjacent dendritic shafts (black). The amplitude distributions and mean amplitudes were unaffected by i.p. A/C administration. Mann-Whitney U test: P = 0.60 (U = 9.5). c , Number of principal components (PCs) accounting for 50% of the total variance across cells (n = 21 cell assemblies from 5 mice). Friedman test (χ² = 49.4, P = 4.8 × 10⁻¹⁰ ) followed by pairwise Wilcoxon signed-rank tests with Bonferroni correction: mobile vs immobile awake, P = 0.23; mobile vs immobile Interm-C, P = 5.7 × 10⁻⁴ ; immobile Interm-C vs State-C, P = 0.077; immobile awake vs immobile Interm-C, P = 8.6 × 10⁻⁵ . d , Participation ratio (PR) for mobile and immobile awake phases before A/C, and for mobile, immobile Interm-C, and State-C after i.p. A/C administration (n = 21 cell assemblies from 5 SynC@FPC mice). Friedman test (χ² = 29.2, P = 7.1 × 10⁻⁶ ) followed by pairwise Wilcoxon signed-rank tests with Bonferroni correction: mobile vs immobile awake, P = 0.029; mobile vs immobile Interm-C, P = 3.3 × 10⁻³ ; immobile Interm-C vs State-C, P = 0.61; immobile awake vs immobile Interm-C, P = 5.1 × 10⁻⁴ .

Article Snippet: The A/C heterodimerizer (Takara Bio) was dissolved in N,N-dimethylacetamide (Fujifilm) at 20 mg mL−1 and diluted in propylene glycol (Nacalai Tesque) to 2 mg mL−1.

Techniques: Isolation, MANN-WHITNEY

Journal: eLife

Article Title: Glucose-stimulated KIF5B-driven microtubule sliding organizes microtubule networks in mouse pancreatic β cells

doi: 10.7554/eLife.89596

Figure Lengend Snippet: ( A ) Schematic of kinesin-1 (KIF5) and the dominant-negative (KIFDN) and heterodimerization strategy. Top schematic shows full-length KIF5s, consisting of the motor domain, stalk coil-coil domain, and the tail. Three constructs utilized here include (1) The KIF5C motor domain tagged with a blue fluorescent protein (BFP) and the FKBP-rapamycin binding (FRB) for heterodimerization; (2) KIFDN wt construct with KIF5B tail domain tagged with the mCherry fluorescent protein and the FKBP for heterodimerization. (3) KIFDN mut construct is the same as (2) but features a set of point mutations (magenta) making the ATP-independent MT-binding domain unable to bind MT lattice. ( B ) Quantification of MT sliding in FRAP assay in cells subjected to DN construct expression and heterodimerization. MT displacement is shown as the area of MTs displaced into the bleached area after 5 min of recovery. See representative data ( C–G ). N=5–25 per condition. One-way ANOVA test was performed for statistical significance (p-value<0.0001; ns, nonsignificant). ( C–G” ) Frames from representative FRAP live-cell imaging sequences of MIN6 cells expressing mEmerald-tubulin. Inverted grayscale images of maximum intensity projections over 1-µm-thick stacks by spinning disk confocal microscopy. (C1–G1) The first frame after photobleaching. (C2–G2) A frame 5 min (300 s) after photobleaching. Light-blue dotted lines indicate the edges of the photobleached areas. Red arrows indicate MTs displaced into the bleached area. Scale bars, 5 µm. (C3–G3) Schematics of experimental manipulation: green represents MTs, blue represents endogenous KIF5B, magenta represents KIFDN wt , purple represents KIFDN mut , gray represents KIF5C motor, orange bracket represents heterodimerizing agent (rap, rapalog). Conditions: (C1–C3) Untreated control. Only endogenous KIF5B is present. (D1–D3) KIFDN wt overexpression. Endogenous KIF5B is unable to bind MTs. (E1–E3) KIFDN mut overexpression. It does not bind MTs and does not interfere with endogenous KIF5B. ( C – E , ‘KFDN FRAP’) (F1–F3) KIFDN wt and KIF5C motor overexpression plus rapalog treatment. Heterodimerization creates a large pool of motors capable of MT sliding. ( G–G” ) KIFDN mut and KIF5C motor overexpression plus rapalog treatment. Heterodimerization creates a large pool of the motor nonfunctional in MT sliding ( F – G , ‘KFDN + motor FRAP’).

Article Snippet: Chemical compound, drug , A/C Heterodimerizing Drug , Takara Bio Inc , Cat#: 635056 , Final concentration (25 μm).

Techniques: Dominant Negative Mutation, Construct, Binding Assay, FRAP Assay, Expressing, Live Cell Imaging, Confocal Microscopy, Control, Over Expression

Journal: bioRxiv

Article Title: The axonal ER couples translation and secretion machineries for local delivery of axonal transmembrane proteins to promote axonal development

doi: 10.1101/2025.09.09.674816

Figure Lengend Snippet: (A-B) Representative images of DIV9-10 axons expressing SEC23A together with SEC24D, SEC31A or SEC16B (A). Quantification of colocalization between SEC23A and SEC24D, SEC31A or SEC16B (B). (C) Representative still and time-lapse from zoomed regions showing the tight association between SEC23A and the axonal ER labelled with SEC61β. Images were acquired at 1-second intervals for 80 seconds. See Video S4. (D-F) Schematic showing the Strep-SBP heterodimerization system using Strep-KIFC1 and SBP-RTN4 to relocate axonal ER to the soma (D). Representative images of SEC23A and RTN4 in DIV10 neurons in the absence (control) or presence of KIFC1 motor (pulled) (E). Quantification of SEC23A number from conditions in (E) in 100 μm axons (F). (G-J) Schematic showing the FKBP-FRB heterodimerization system using KIFC1-FRB and FKBP-SEC23A to relocate axonal ERES components to the soma (G). Representative images of SEC23A treated with ethanol (vehicle, control) or Rapalog for 16h (H). Representative images of RUSH-SYT1 cargos in neurons treated as in (H) for 16h, prior biotin addition for 4h in the presence of BFA and BDNF (I). Quantification of RUSH-SYT1 cargo number from conditions in (I) in 100 μm axons (J). (K-L) Distribution of SEC23A in the axon in control BSA and after 30 min BDNF stimulation (K). Quantification of SEC23A puncta number per 100 μm axon (L). (M-O) Schematic showing the timeline of different treatments before fixation (M). Representative images of DIV10-11 neurons expressing RUSH-SYT1 after 4h biotin release, 4h biotin release and pre-treated with BFA alone, or 4h biotin release together with BFA and BDNF. Live cell surface labeling for HA-tag inserted in the luminal/extracellular domain of RUSH-SYT1, allows for the visualization of SYT1 fusion with the PM (N). Quantification of the percentage of released cargos fusing with the PM (O). See also related Figure S5. Data are presented as box-and-whisker plots in (B, F, J, L, O). Individual data points each represent a neuron, and each color per bar represents an independent experiment. *p < 0.05, **p < 0.01, *** p < 0.001, ****p<0.001 comparing conditions using unpaired t-test in (F, J, L) or ordinary one-way ANOVA test followed by Tukey’s multiple comparisons test in (O). Scale bars represent 10 μm in (A, E, H, I, K, N), 5 μm in the overview image, and 1 μm in cropped images in (C).

Article Snippet: Other reagents used in this study were: Puromycin dihydrochloride (Sigma-Aldrich, Cat#P8833), Anisomycin (Sigma-Aldrich, Cat#9789), Lipofectamine 2000 (Thermo Fisher Scientific, Cat#1639722), Fluoromount-G Mounting Medium (ThermoFisher Scientific, Cat#00-4958-02), anhydrous-DMSO (Thermo Fisher Scientific, Cat#D12345), biotin (Sigma-Aldrich, Cat#B4501), biotin-phenol (Iris Biotech, Cat#LS.3500), H 2 O 2 (Sigma-Aldrich, Cat#H1009), Sodium Azide (Merck, Cat#K43547688), Sodium L-ascorbate (Sigma, Cat#0000374819), Trolox (Sigma, Cat#238813), rapalog (A/C Heterodimerizer) (Takara Bio, Cat#635057), NeutrAvidin (Thermo Fisher Scientific, Cat#31000), Brefeldin A (Sigma-Aldrich, Cat#B5936), NAD+ (Sigma-Aldrich, Cat# 10127965001), Mix-n-Stain CF640R antibody labeling kit (Biotium, Cat#92245), Duolink® In Situ Detection Reagents FarRed (Sigma-Aldrich, Cat#DUO92013), Polyethylenimine (PEI MAX; Polysciences, Cat#24765), and Triton-X-100 (Sigma, Cat#93433).

Techniques: Expressing, Control, Labeling, Whisker Assay