Journal: Frontiers in Immunology

Article Title: Reversing T cell dysfunction in a novel in vitro model of T cell exhaustion reveals differential roles of RASA2

doi: 10.3389/fimmu.2026.1509926

Figure Lengend Snippet: Chronic in vitro stimulation of T cells results in an exhaustion phenotype. (A) Schematic illustrating the in vitro exhaustion protocol developed using chronic stimulation with soluble aCD3/aCD28 Immunocult™ activator in the presence of IL-2. Unstimulated T cells receive 6 repeated 48–72-hour stimulations to generate exhausted (Tex) cells. Following the first stimulation, a proportion of T cells are rested in IL-2 media for the remainder of the exhaustion protocol as a functional single stimulated T cell (Ts) control. Following the 6 th stimulation for the Tex cells, both Tex and Ts populations are characterized by immunophenotyping. Subsequently, an endpoint Immunocult ™ stimulation is performed on both Tex and Ts cells in parallel, with further phenotypic and functional characterization using immunophenotyping by flow cytometry and cytokine secretion using MSD being conducted after 48 hours. (B) Representative dot plots and quantification of %PD-1 + Tim-3 + in Tex and Ts CD8 + (n=13 donors) and CD4 + (n=8 donors) T cells at the end of the exhaustion protocol. (C) Quantification (%) of 4-1BB + expression assessed by flow cytometry and IFN-γ, TNF-α, and IL-2 secretion (pg/mL) assessed by MSD in CD8 + (n= 7 donors) and CD4 + (n=7–8 donors) Tex and Ts following endpoint Immunocult™ stimulation. Points represent individual donors. Statistical analysis was performed using a Wilcoxon test; *p<0.05, **p<0.01, ***p<0.005.

Article Snippet: Antibodies ( ) were prepared at a 2X concentration in eBioscienceTM Flow Cytometry Staining Buffer (FACS buffer; Thermo Fisher, #00-4222-26) and added to the cell directly after blocking, for 20 minutes at room temperature protected from light.

Techniques: In Vitro, Functional Assay, Control, Flow Cytometry, Expressing

Journal: Frontiers in Immunology

Article Title: Hyaluronic acid−CD44 signaling from decidual stromal cells orchestrates dNK1 differentiation and immune tolerance in early pregnancy

doi: 10.3389/fimmu.2026.1777567

Figure Lengend Snippet: HA/CD44 axis promotes NK92MI tissue residency. (A) Analysis of HAS2 expression levels across different cell subpopulations in normal decidual tissue based on scRNA-seq data ( n = 11). (B) The expression of HAS2 in human decidual tissue (scale bars, 100 μm) by immunohistochemistry ( n = 10 per group). (C) Confirmation of HAS2KD in DSCs by western blot ( n = 3 per group). (D) HA concentration in DSC supernatant ( n = 6 per group). (E) The residency of NK92MI cells in DSCs (scale bars, 50 μm) after co-culture with HAS2KD DSCs ( n = 9 per group). (F, G) The expression of CD49a and adhesion molecules ( ICAM-1, VCAM-1 , and ITGAX ) in NK92MI cells after co-culture with HAS2KD DSCs ( n = 6 per group). (H) CD49a expression in NK92MI cells co−cultured with control DSCs or HAS2KD DSCs, with or without exogenous HMW−HA ( n = 6 per group). (I) CD49a expression in NK92MI cells from DSC-NK co-cultures treated with exogenous HMW-HA after CD44 blocking ( n = 6 per group). (J, K) The expression of CD49a and adhesion molecules ( ICAM-1, VCAM-1 , and ITGAX ) in NK92MI cells co−cultured with control DSCs or HAS2KD DSCs, with or without CD44−blocking antibody. Data are expressed as mean ± SD; * P < 0.05; ** P < 0.01; ns, not significant. EP, early pregnancy; SA, spontaneous abortion.

Article Snippet: To determine the signaling pathways involved, we used an anti-CD44 blocking antibody (30 μg/mL; BioxCell, BE0262) to block CD44, and IWP-2 (50 μM; MedChemExpress, HY-13912) to inhibit the canonical Wnt pathway.

Techniques: Expressing, Immunohistochemistry, Western Blot, Concentration Assay, Co-Culture Assay, Cell Culture, Control, Blocking Assay

Journal: Frontiers in Immunology

Article Title: Hyaluronic acid−CD44 signaling from decidual stromal cells orchestrates dNK1 differentiation and immune tolerance in early pregnancy

doi: 10.3389/fimmu.2026.1777567

Figure Lengend Snippet: DSCs ameliorate NK92MI cell cytotoxicity by modulating CD44 subpopulations via the HA/CD44 axis. (A) The proportion of the CD44 high subpopulation in NK92MI cells after co-culture with DSCs ( n = 6 per group). (B) The expression of cytokines (GZMB, TNF-α, IFN-γ, TGF-β1 and IL-10) in NK92MI cells detected by flow cytometry after co-culture with DSCs ( n = 5 per group). (C) The viability of K562 cells determined after exposure to NK cells previously co-cultured with DSCs ( n = 6 per group). (D, E) The proportion of the CD44 high subpopulation and the expression of effector molecules (GZMB, TNF−α, IFN−γ, TGF−β1 and IL−10) in NK92MI cells co−cultured with control DSCs or HAS2KD DSCs, with or without CD44−blocking antibody ( n = 6 per group). (F, G) The proportion of the CD44 high subpopulation in NK92MI cells co−cultured with control DSCs, HAS2KD DSCs, or DSCs plus CD44−blocking antibody after the addition of exogenous HMW−HA ( n = 5 per group). Data are expressed as mean ± SD; ** P < 0.01; ns, not significant.

Article Snippet: To determine the signaling pathways involved, we used an anti-CD44 blocking antibody (30 μg/mL; BioxCell, BE0262) to block CD44, and IWP-2 (50 μM; MedChemExpress, HY-13912) to inhibit the canonical Wnt pathway.

Techniques: Co-Culture Assay, Expressing, Flow Cytometry, Cell Culture, Control, Blocking Assay

Journal: Frontiers in Immunology

Article Title: Hyaluronic acid−CD44 signaling from decidual stromal cells orchestrates dNK1 differentiation and immune tolerance in early pregnancy

doi: 10.3389/fimmu.2026.1777567

Figure Lengend Snippet: DSCs ameliorate NK cell cytotoxicity by regulating the proportion of CD44 high and CD44 low subpopulations via the HA/CD44 axis. (A) The proportion of dNK1, dNK2, and dNK3 subpopulations in dNK cells from individuals with normal pregnancies and SA ( n = 19 per group). (B) The viability of K562 cells after co-culture with total dNK cells from individuals of normal pregnancies and SA ( n = 9 per group). (C) UMAP plot of scRNA-seq data from normal decidual tissues showing elevated CD44 expression in the dNK2 and dNK3 subpopulation ( n = 11). (D) The proportion of the CD44 high subpopulation within the three subpopulations in individuals with normal pregnancies ( n = 6). (E) The proportion of the CD44 high subpopulation in total dNK cells and the three subpopulations between individuals from normal pregnancies and SA ( n = 6 per group). Data are expressed as mean ± SD; ** P < 0.01; ns, not significant. EP, early pregnancy; SA, spontaneous abortion.

Article Snippet: To determine the signaling pathways involved, we used an anti-CD44 blocking antibody (30 μg/mL; BioxCell, BE0262) to block CD44, and IWP-2 (50 μM; MedChemExpress, HY-13912) to inhibit the canonical Wnt pathway.

Techniques: Co-Culture Assay, Expressing

Journal: Frontiers in Immunology

Article Title: Hyaluronic acid−CD44 signaling from decidual stromal cells orchestrates dNK1 differentiation and immune tolerance in early pregnancy

doi: 10.3389/fimmu.2026.1777567

Figure Lengend Snippet: HA-induced activation of the canonical Wnt pathway upregulates FOSL2 to expand the dNK1-like subpopulation. (A) Analysis of transcriptional activity and expression levels of transcription factors in three dNK subsets using scRNA-seq data from normal decidual tissue ( n = 11). (B) Transcriptional activity and expression levels of FOSL2 in decidual tissues from normal pregnancy ( n = 5) and RSA groups ( n = 3). (C) Changes in NK92MI cells of the five transcription factors ( STAT3, MAFB, HES1, FOSL2, ETV5 ) characterized by high transcriptional activity and expression in the dNK1 subset following HMW-HA treatment, as determined by RT−qPCR ( n = 6 per group). (D) FOSL2 expression in NK92MI cells following HMW-HA treatment, assessed by WB ( n = 3 per group). (E) Wnt1 and β-catenin protein expression in NK92MI cells treated with or without HMW-HA in the presence or absence of CD44 blockade ( n = 3 per group). (F) FOSL2 expression in HA−treated NK92MI cells after addition of CD44−blocking antibody or the Wnt pathway inhibitor IWP−2 to the culture system ( n = 3 per group). Data are expressed as mean ± SD; * P < 0.05; ** P < 0.01; ns, not significant. EP, early pregnancy; SA, spontaneous abortion; RSA, recurrent spontaneous abortion.

Article Snippet: To determine the signaling pathways involved, we used an anti-CD44 blocking antibody (30 μg/mL; BioxCell, BE0262) to block CD44, and IWP-2 (50 μM; MedChemExpress, HY-13912) to inhibit the canonical Wnt pathway.

Techniques: Activation Assay, Activity Assay, Expressing, Quantitative RT-PCR, Blocking Assay

Journal: Oncogene

Article Title: ECM1 regulates tumor metastasis and CSC-like property through stabilization of β-catenin.

doi: 10.1038/onc.2015.54

Figure Lengend Snippet: Figure 1. Effect of ECM1 on cell migration and invasion in breast cancer cells. (a) ELISA showing circulating levels of ECM1 in plasma from breast cancer patients. Error bars represent mean ± s.d. of all experiments (**Po0.005). (b and c) Each cell was plated on transwell plates for the migration assay (b) or invasion assay (c). The number of cells on the bottom layer was counted at 24 h after incubation. In the case of MDA-MB-231, cells were counted at 18 h. Each bar graph represents fold-increase compared with control cells. Error bars represent mean ± s.d. of triplicate experiments (*Po0.05, **Po0.005). (d) Lung metastatic nodules driven by tail vein injection of MDA-MB-231 control shRNA (shC, n = 5) and ECM1 shRNA (shE, n = 4) cells were counted with the naked eye. Bar graph represents the number of nodules on the lung surface. Error bars represent mean ± s.d. of all experiments (*Po0.05). (e) Representative hematoxylin and eosin staining of the tumor margin showed local invasiveness.

Article Snippet: Two types of anti-ECM1 antibodies (N-17 and P-19), which blocked function of extracellular ECM1 were obtained from Santa Cruz Biotechnology.

Techniques: Migration, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Invasion Assay, Incubation, Control, Injection, shRNA, Staining

Journal: Oncogene

Article Title: ECM1 regulates tumor metastasis and CSC-like property through stabilization of β-catenin.

doi: 10.1038/onc.2015.54

Figure Lengend Snippet: Figure 2. Effect of ECM1 on sphere formation and drug resistance in breast cancer cells. (a) Cells were seeded on polyhema-coated plates with bFGF, EGF and B27. After 7 days, sphere-forming rates were measured. Error bars represent mean ± s.d. of triplicate experiments (*Po0.05, **Po0.005). Scale bar = 100 μm. (b) All cells were treated with doxorubicin. After a further 48 h, cell viability was analyzed using MTT assays. Error bars represent mean ± s.d. of triplicate experiments (*Po0.05, **Po0.005).

Article Snippet: Two types of anti-ECM1 antibodies (N-17 and P-19), which blocked function of extracellular ECM1 were obtained from Santa Cruz Biotechnology.

Techniques:

Journal: Oncogene

Article Title: ECM1 regulates tumor metastasis and CSC-like property through stabilization of β-catenin.

doi: 10.1038/onc.2015.54

Figure Lengend Snippet: Figure 3. ECM1 modulates the gene expression related to EMT progression and CSC maintenance. (a–c) mRNA levels of each gene were determined by real-time PCR using specific primers (VM = Vimentin, E-cad = E-cadherin). Error bars represent mean ± s.d. of triplicate experiments (*Po0.05, **Po0.005, ***Po0.0005). (d) Each cell lysate was analyzed by western blotting using ZEB1, Vimentin, E-cadherin and actin antibodies. (e) At 24 h after cell seeding, all cells were treated with rhECM1 (200 ng/ml). After further 48 h of treatment, cell lysates were prepared for western blotting with indicated antibodies.

Article Snippet: Two types of anti-ECM1 antibodies (N-17 and P-19), which blocked function of extracellular ECM1 were obtained from Santa Cruz Biotechnology.

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Western Blot

Journal: Oncogene

Article Title: ECM1 regulates tumor metastasis and CSC-like property through stabilization of β-catenin.

doi: 10.1038/onc.2015.54

Figure Lengend Snippet: Figure 4. ECM1 upregulates β-catenin expression level. (a) Each cell lysate was analyzed by western blotting using β-catenin and actin antibodies. (b and c) At 24 h after cell seeding, all cells were treated with rhECM1 (200 ng/ml) or anti-ECM1 antibodies (5 μg/ml). After a further 48 h, cell lysates were prepared for western blotting with indicated antibodies. (d and e) β-catenin-dependent transcriptional activities were measured using TOPFLASH reporter (d) or Axin2 and Cyclin D1 reporters (e). Error bars represent mean ± s.d. of triplicate experiments (*Po0.05, **Po0.005, ***Po0.005). (f) ZEB1 promoter activity was measured with dual luciferase assay. Error bars represent mean ± s.d. of triplicate experiments (*Po0.05, **Po0.005, ***Po0.005).

Article Snippet: Two types of anti-ECM1 antibodies (N-17 and P-19), which blocked function of extracellular ECM1 were obtained from Santa Cruz Biotechnology.

Techniques: Expressing, Western Blot, Activity Assay, Luciferase

Journal: Oncogene

Article Title: ECM1 regulates tumor metastasis and CSC-like property through stabilization of β-catenin.

doi: 10.1038/onc.2015.54

Figure Lengend Snippet: Figure 5. ECM1 stabilizes β-catenin protein and induces the accumulation of nuclear β-catenin via MUC1. (a) Each cell was treated with CHX at 100 μg/ml. The cell lysates were obtained at the indicated time points and subjected to western blot analysis with the indicated antibodies. The intensities of the bands were quantified using 1DScan software and plotted as time versus the ratio of β-catenin/Actin intensity. Error bars represent mean ± s.d. of triplicate experiments (*P o0.05, **Po0.005). (b) Each cell was fractionated into cytoplasmic and nuclear fractions. The lysates were analyzed by western blotting with the indicated antibodies. (c) Each cell lysate was analyzed by western blotting using indicated antibodies. (d) Total cell lysates were incubated with normal IgG or MUC1 CT antibodies, and immunoprecipitates were then analyzed on western blots. (e) At 24 h after cell seeding, each cell was treated with galectin-3 and MUC1 siRNA. After a further 48 h, cell lysates were prepared for western blotting with indicated antibodies. (f) Each cell was transfected with galecin-3 or MUC1 siRNA and then β-catenin- dependent transcriptional activities were examined using TOPFLASH reporter. Error bars represent mean ± s.d. of triplicate experiments (**Po0.005, ***Po0.0005).

Article Snippet: Two types of anti-ECM1 antibodies (N-17 and P-19), which blocked function of extracellular ECM1 were obtained from Santa Cruz Biotechnology.

Techniques: Western Blot, Software, Incubation, Transfection

Journal: Oncogene

Article Title: ECM1 regulates tumor metastasis and CSC-like property through stabilization of β-catenin.

doi: 10.1038/onc.2015.54

Figure Lengend Snippet: Figure 6. Forced expression of β-catenin causes the modulation of gene expression toward EMT progression and CSC maintenance. (a and b) The cells were transfected with pcDNA-β-catenin (a) or β-catenin siRNA (b) and then mRNA levels of each gene were determined by real-time PCR using specific primers (VM = Vimentin, E-cad = E-cadherin). Error bars represent mean ± s.d. of triplicate experiments (*Po0.05, **Po0.005, ***Po0.0005). (c) Each cell was transfected with pcDNA-β-catenin or β-catenin siRNA and then each cell lysate was subjected to western blot analysis using indicated antibodies. (d) Schematic model showing the role of ECM1 in regulation of EMT and CSC capacity.

Article Snippet: Two types of anti-ECM1 antibodies (N-17 and P-19), which blocked function of extracellular ECM1 were obtained from Santa Cruz Biotechnology.

Techniques: Expressing, Gene Expression, Transfection, Real-time Polymerase Chain Reaction, Western Blot

Journal: eLife

Article Title: Site-directed MT1-MMP trafficking and surface insertion regulate AChR clustering and remodeling at developing NMJs

doi: 10.7554/eLife.54379

Figure Lengend Snippet: ( A ) Representative images showing the spatial localization of endogenous MT1-MMP in perforations of aneural AChR clusters (arrows) in fixed Xenopus muscle cultures. ( B ) Representative images showing the extent of fluorescent gelatin degradation in area covered by MT1-MMP-mCherry-overexpressing (MT1-mCherry) muscle cells in the presence or absence of MMP inhibitors BB-94 or BB-2516. 8-bit pseudo-color images highlight the relative fluorescence intensity of AChR clusters in different conditions. ( C ) Quantification on the effects of MT1-MMP-mCherry overexpression on the formation of AChR top and bottom clusters in response to BB-94 or BB-2516 treatment. n = 163 (Control), 123 (MT1-mCherry), 119 (MT1-mCherry + BB-94), and 62 (MT1-mCherry + BB-2516) muscle cells from 4 independent experiments. ( D ) Quantification on the effects of MT1-MMP-mCherry overexpression on the extent of fluorescent gelatin degradation and the intensity of aneural AChR clusters in response to BB-94 or BB-2516 treatment. For gelatin intensity measurement: n = 30 (Control), 39 (MT1-mCherry), 33 (MT1-mCherry + BB-94), and 31 (MT1-mCherry + BB-2516) muscle cells from 3 independent experiments. For AChR intensity measurement: n = 14 (Control), 19 (MT1-mCherry), 21 (MT1-mCherry + BB-94), and 16 (MT1-mCherry + BB-2516) muscle cells from 3 independent experiments. Scale bars represent 10 μm. Data are represented as mean ± SEM. One-way ANOVA with Dunnett’s multiple comparisons test ( C ), one-way ANOVA with Turkey’s multiple comparisons test ( D ), *, **, **** represent p≤0.05, 0.01, and 0.0001 respectively.

Article Snippet: After blocking with 2% BSA and 0.2% Triton X-100 at room temperature for 2 hr, the sections were labeled with primary antibody against MT1-MMP (1:100; Santa Cruz Biotechnology, sc-373908 and 1:100; Millipore, MAB3328), neurofilament NF200 (1:500; Sigma, N4142), and synaptophysin (1:1000; Abcam, ab32127) for 2 days at 4°C.

Techniques: Fluorescence, Over Expression

Journal: eLife

Article Title: Site-directed MT1-MMP trafficking and surface insertion regulate AChR clustering and remodeling at developing NMJs

doi: 10.7554/eLife.54379

Figure Lengend Snippet: ( A ) Representative images showing the intracellular trafficking of MT1-MMP vesicles in a cultured muscle cell expressing both MT1-MMP-mCherry (MT1-mCherry) and EB1-GFP. The time-lapse series of two regions (i and ii) outlined in the merge image showed (i) the search-and-capture of MT1-mCherry vesicles (arrows) by an EB1-GFP comet (arrowheads); and (ii) the bidirectional movement of MT1-mCherry vesicles (arrows) along the microtubules (arrowheads). ( B ) Kymographs showing the spatiotemporal correlation between MT1-mCherry and EB1-GFP signals along K1 and K2 lines indicated in the merge image. The maximal projection of MT1-mCherry and EB1-GFP signals was constructed from 40 frames in a 40 s time-lapse series. Arrows indicate the examples of lateral displacement of initially stationary MT1-mCherry vesicles after EB1-GFP comets had passed through. ( C ) Representative TIRF-FRAP images showing the local capturing of MT1-mCherry vesicles at AChR clusters. After photobleaching, the recovery of MT1-mCherry signals and their trajectories in two regions of interest indicate (i) MT1-mCherry vesicles (arrows) were transported to and captured at the perforation of AChR clusters; and (ii) two groups of MT1-mCherry vesicles (red and green arrows) were transported to and captured at the same site of AChR cluster periphery over a period of 120 s. ( D ) Kymographs showing the spatiotemporal capture of MT1-mCherry vesicles at AChR clusters. Two kymographs were constructed from 120 time-lapse images along K3 and K4 lines, as indicated in the merge image (top panels in C). Arrowheads and arrows indicate the sites of MT1-mCherry capture at the perforated and peripheral regions of AChR clusters, respectively. Scale bars represent 10 μm, unless stated otherwise.

Article Snippet: After blocking with 2% BSA and 0.2% Triton X-100 at room temperature for 2 hr, the sections were labeled with primary antibody against MT1-MMP (1:100; Santa Cruz Biotechnology, sc-373908 and 1:100; Millipore, MAB3328), neurofilament NF200 (1:500; Sigma, N4142), and synaptophysin (1:1000; Abcam, ab32127) for 2 days at 4°C.

Techniques: Cell Culture, Expressing, Construct

Journal: eLife

Article Title: Site-directed MT1-MMP trafficking and surface insertion regulate AChR clustering and remodeling at developing NMJs

doi: 10.7554/eLife.54379

Figure Lengend Snippet: Representative images showing the localization of some endogenous MT1-MMP signals at the vesicular compartment (arrows), as visualized by vesicle-associated membrane protein 1 (VAMP1). For clarity, two regions were shown in the 2X magnified view. Scale bar represents 10 μm.

Article Snippet: After blocking with 2% BSA and 0.2% Triton X-100 at room temperature for 2 hr, the sections were labeled with primary antibody against MT1-MMP (1:100; Santa Cruz Biotechnology, sc-373908 and 1:100; Millipore, MAB3328), neurofilament NF200 (1:500; Sigma, N4142), and synaptophysin (1:1000; Abcam, ab32127) for 2 days at 4°C.

Techniques:

Journal: eLife

Article Title: Site-directed MT1-MMP trafficking and surface insertion regulate AChR clustering and remodeling at developing NMJs

doi: 10.7554/eLife.54379

Figure Lengend Snippet: ( A ) Representative images showing the effects on aneural AChR clustering in cultured muscle cells over-expressing different levels of MT1-MMP-pHluorin (MT1-pHluorin). Arrows indicate the spatial localization of MT1-pHluroin at perforated regions of AChR clusters. ( B ) Quantification on the effects on aneural AChR cluster formation in cultured muscle cells with different levels of MT1-pHluorin over-expression. Cultured muscle cells with average MT1-pHluorin intensity above the cutoff value of >500 (arbitrary unit) were classified as high expression. n = 150 (Control), 32 (Low MT1-pHluorin level), and 15 (High MT1-pHluorin level) muscle cells from 3 independent experiments. ( C ) Representative sets of images showing the change in MT1-pHluorin fluorescence intensity in response to a sequential switching of various culture media with different pH levels. Arrows indicate the spatial localization of MT1-pHluroin signals in the perforated region of AChR clusters. 8-bit pseudo-color images highlight the change in the relative intensity of MT1-pHluorin. ( D ) Representative sets of time-lapse images showing the extent of AChR cluster remodeling in muscle cells with different MT1-pHluorin expression levels. 8-bit pseudo-color images in the insets highlight the change in the relative intensity of AChR clusters. The percentage values indicate the relative fluorescence intensity of AChR clusters after 24 hr. ( E ) A scatter plot analysis showing the correlation between MT1-pHluorin intensity and the percentage change in AChR intensity over 24 hr in cells with different MT1-pHluorin expression levels. The black line indicates a linear correlation. R = −0.7188, p=0.0056. n = 13 from 3 independent experiments. ( F ) Representative TIRF-FRAP images showing the surface insertion of MT1-pHluorin at AChR clusters in cultured muscle cells. Arrows and arrowheads indicate the spatial insertion of MT1-pHluorin at the perforation and periphery of AChR clusters, respectively. ( G ) Kymographs showing the spatiotemporal insertion of MT1-pHluorin at AChR clusters. Two kymographs were constructed from 180 time-lapse images along the lines of K1 and K2, as indicated in the merge image (left panel). Arrows indicate some newly inserted MT1-pHluorin that were relatively stable, while arrowheads indicate that some were dispersed shortly after surface insertion. ( H ) Quantification on the number of events of MT1-pHluorin surface insertion per unit area (μm 2 ) of aneural AChR clusters over the entire time-lapse duration. n = 10 (Control) and 10 (CLASP-MO) muscle cells from 3 independent experiments. Scale bars represent 10 μm, unless otherwise specified. Data are represented as mean ± SEM. One-way ANOVA with Bonferroni’s multiple comparisons test (B), Student’s t-test ( H ), **, *** represent p≤0.01 and 0.001 respectively. n.s.: non-significant.

Article Snippet: After blocking with 2% BSA and 0.2% Triton X-100 at room temperature for 2 hr, the sections were labeled with primary antibody against MT1-MMP (1:100; Santa Cruz Biotechnology, sc-373908 and 1:100; Millipore, MAB3328), neurofilament NF200 (1:500; Sigma, N4142), and synaptophysin (1:1000; Abcam, ab32127) for 2 days at 4°C.

Techniques: Cell Culture, Expressing, Over Expression, Fluorescence, Construct

Journal: eLife

Article Title: Site-directed MT1-MMP trafficking and surface insertion regulate AChR clustering and remodeling at developing NMJs

doi: 10.7554/eLife.54379

Figure Lengend Snippet: ( A ) Representative images showing the increased signals of endogenous MT1-MMP at the perforated regions of aneural AChR clusters after 8 hr agrin stimulation. ( B ) Quantification on the time-dependent increase in the localization of MT1-MMP signals at aneural AChR clusters in cultured muscle cells upon agrin treatment. n = 67 (Control, 4 hr), 92 (Agrin, 4 hr), 118 (Control, 8 hr), and 93 (Agrin, 8 hr) muscle cells from 3 independent experiments. Scale bar represents 10 μm. Data are represented as mean ± SEM. Student’s t-test, **, *** represent p≤0.01 and 0.001 respectively.

Article Snippet: After blocking with 2% BSA and 0.2% Triton X-100 at room temperature for 2 hr, the sections were labeled with primary antibody against MT1-MMP (1:100; Santa Cruz Biotechnology, sc-373908 and 1:100; Millipore, MAB3328), neurofilament NF200 (1:500; Sigma, N4142), and synaptophysin (1:1000; Abcam, ab32127) for 2 days at 4°C.

Techniques: Cell Culture

Journal: eLife

Article Title: Site-directed MT1-MMP trafficking and surface insertion regulate AChR clustering and remodeling at developing NMJs

doi: 10.7554/eLife.54379

Figure Lengend Snippet: Western blot analysis showing the reduced endogenous MT1-MMP protein level in Xenopus embryos injected with MT1-MMP-MO. α-tubulin was used as a loading control.

Article Snippet: After blocking with 2% BSA and 0.2% Triton X-100 at room temperature for 2 hr, the sections were labeled with primary antibody against MT1-MMP (1:100; Santa Cruz Biotechnology, sc-373908 and 1:100; Millipore, MAB3328), neurofilament NF200 (1:500; Sigma, N4142), and synaptophysin (1:1000; Abcam, ab32127) for 2 days at 4°C.

Techniques: Western Blot, Injection

Journal: eLife

Article Title: Site-directed MT1-MMP trafficking and surface insertion regulate AChR clustering and remodeling at developing NMJs

doi: 10.7554/eLife.54379

Figure Lengend Snippet: ( A ) Representative images showing the localization of pre-existing and newly inserted AChR clusters at nerve-muscle contacts (arrows) in control co-cultures (WT (M)) and in the chimeric co-cultures of wild-type neurons and muscle cells with control morpholino (Control MO (M)), MT1-MMP-MO (MT1-MO (M)), or MT1-MMP-mCherry and MT1-MMP-MO (MT1-mCherry + MT1-MO (M)) muscles. Fluorescent dextran indicates the muscle cells derived from MO-injected embryos. 8-bit pseudo-color images highlight the relative fluorescence intensity of pre-existing and newly inserted AChR signals. ( B ) Quantification on the percentage of nerve-muscle contacts with AChR clusters in control nerve-muscle co-cultures and in different chimeric co-cultures. n = 127 (WT), 120 (Control MO), 146 (MT1-MO), or 34 (MT1-mCherry + MT1-MO) nerve-muscle pairs from 3 independent experiments. ( C ) Quantification on the intensity of pre-existing AChR signals per unit length of nerve-muscle contacts in control co-cultures and in different chimeric co-cultures. n = 29 (WT), 30 (Control MO), 32 (MT1-MO), or 23 (MT1-mCherry + MT1-MO) nerve-muscle pairs from 4 independent experiments. ( D ) Quantification on the intensity of newly inserted AChR signals per unit length of nerve-muscle contacts in control co-cultures and in different chimeric co-cultures. n = 24 (WT), 23 (Control MO), 25 (MT1-MO), or 19 (MT1-mCherry + MT1-MO) nerve-muscle pairs from 3 independent experiments. Scale bar represents 10 μm. Data are represented as mean ± SEM. One-way ANOVA with Sidak’s multiple comparisons test, *, **, *** represent p≤0.05, 0.01, and 0.001 respectively. n.s.: non-significant.

Article Snippet: After blocking with 2% BSA and 0.2% Triton X-100 at room temperature for 2 hr, the sections were labeled with primary antibody against MT1-MMP (1:100; Santa Cruz Biotechnology, sc-373908 and 1:100; Millipore, MAB3328), neurofilament NF200 (1:500; Sigma, N4142), and synaptophysin (1:1000; Abcam, ab32127) for 2 days at 4°C.

Techniques: Derivative Assay, Injection, Fluorescence

Journal: eLife

Article Title: Site-directed MT1-MMP trafficking and surface insertion regulate AChR clustering and remodeling at developing NMJs

doi: 10.7554/eLife.54379

Figure Lengend Snippet: ( A ) A schematic diagram illustrating the use of laser-based photobleaching approach to differentially identify the contribution of aneural AChR clusters and diffuse AChRs for the assembly of nerve-induced synaptic AChR clusters. ( B ) Representative images showing the differential contribution of aneural AChR clusters and diffuse AChRs to nerve-induced synaptic AChR clusters in control co-cultures (WT) and in the chimeric co-cultures of wild-type neurons and muscle cells with control MO or MT1-MMP MO. Yellow boxes (right panels) indicate the region of photobleaching. White boxes show a magnified view of nerve-muscle contacts in 1-d old co-cultures. Dotted lines highlight the periphery of muscle cells. Fluorescent dextran signals in the insets indicate the muscle cells with microinjected MO. 8-bit pseudo-color images highlight the relative fluorescence intensity of AChR signals. Arrows indicate synaptic AChR clusters at nerve-muscle contact sites. Arrowheads indicate the original location of aneural AChR clusters. ( C ) Quantification on the intensity of AChR signals at the nerve-muscle contacts in control co-cultures and in chimeric co-cultures either with or without photobleaching. The control groups without photobleaching indicate the contribution from aneural AChR clusters + diffuse AChRs, n = 15 (WT), 9 (Control MO), and 9 (MT1-MMP MO) nerve-muscle pairs from 3 independent experiments. The experimental groups with photobleaching of aneural AChR clusters indicate the contribution from diffuse AChRs only, n = 23 (WT), 13 (Control MO), and 8 (MT1-MMP MO) nerve-muscle pairs from 3 independent experiments. Scale bars represent 10 μm. Data are represented as mean ± SEM. Two-way ANOVA with Sidak’s multiple comparison test, *, ** represent p≤0.05 and 0.01 respectively.

Article Snippet: After blocking with 2% BSA and 0.2% Triton X-100 at room temperature for 2 hr, the sections were labeled with primary antibody against MT1-MMP (1:100; Santa Cruz Biotechnology, sc-373908 and 1:100; Millipore, MAB3328), neurofilament NF200 (1:500; Sigma, N4142), and synaptophysin (1:1000; Abcam, ab32127) for 2 days at 4°C.

Techniques: Fluorescence

Journal: eLife

Article Title: Site-directed MT1-MMP trafficking and surface insertion regulate AChR clustering and remodeling at developing NMJs

doi: 10.7554/eLife.54379

Figure Lengend Snippet: ( A ) Representative confocal images showing the localization of endogenous MT1-MMP at perforations of synaptic AChR clusters (arrows, top panels) in longitudinal cryosections of adult rat soleus muscles. The specificity of MT1-MMP antibody was verified by pre-incubating anti-MT1-MMP primary antibody with recombinant human MT1-MMP protein (rhMT1-MMP, bottom panels). Neurofilament (NF) was used as a neuronal marker. ( B ) Representative confocal images showing the postsynaptic localization of MT1-MMP at NMJs as demonstrated by the surgical denervation experiment. Staining of AChR, MT1-MMP, and presynaptic marker (NF and synaptophysin (Syn)) was performed using cross sections from adult rat soleus muscles either without (top panels) or with sciatic nerve cut after 4 days (bottom panels). ( C ) Representative confocal images showing aneural versus synaptic AChR clusters in whole-mount diaphragms from wild-type (WT) control and MT1-MMP -/- mice at E13.5 (left panels) and E18.5 (right panels). Whole-mount tissues were stained for AChR and NF. The superimposed 3D reconstruction images were generated by z-stack images using Imaris. Synaptic AChR clusters (yellow) were identified when signals of AChR and NF overlapped with each other, whereas other AChR signals (red) represent aneural AChR clusters. ( D–G ) Quantification on the density of aneural ( D ) versus synaptic ( E ) AChR clusters, the width of end-plate bands ( F ), and the length of axonal branches ( G ) in diaphragm muscles between wild-type and MT1-MMP -/- mouse embryos at E13.5 and E18.5. n = 4 (E13.5, WT) and 5 (E13.5, MT1-MMP -/- ), 5 (E18.5, WT), and 3 (E18.5, MT1-MMP -/- ) embryos from three independent experiments. Scale bars represent 10 μm ( A ) or 100 μm ( C ). Data are represented as mean ± SEM. Two-way ANOVA with Sidak’s multiple comparison test ( D and E ), Student’s t-test ( F and G ), *, **, *** represent p≤0.05, 0.01, and 0.001 respectively. n.s.: non-significant.

Article Snippet: After blocking with 2% BSA and 0.2% Triton X-100 at room temperature for 2 hr, the sections were labeled with primary antibody against MT1-MMP (1:100; Santa Cruz Biotechnology, sc-373908 and 1:100; Millipore, MAB3328), neurofilament NF200 (1:500; Sigma, N4142), and synaptophysin (1:1000; Abcam, ab32127) for 2 days at 4°C.

Techniques: Recombinant, Marker, Staining, Generated

Journal: eLife

Article Title: Site-directed MT1-MMP trafficking and surface insertion regulate AChR clustering and remodeling at developing NMJs

doi: 10.7554/eLife.54379

Figure Lengend Snippet: ( A ) Schematic diagram showing the proposed mechanisms underlying site-directed MT1-MMP trafficking and surface insertion for the regulation of focal ECM degradation and the recruitment of ECM-induced bottom aneural AChR clusters to nerve-induced synaptic clusters at developing NMJs. ( B ) Logical flow diagram proposing key events in the mechanistic role of ECM-induced PLS assembly in MT1-MMP trafficking and surface insertion, and the functional role of MT1-MMP-mediated ECM degradation in postsynaptic development. The relevant data presented in the main figures that support each of the individual proposed events are highlighted in red.

Article Snippet: After blocking with 2% BSA and 0.2% Triton X-100 at room temperature for 2 hr, the sections were labeled with primary antibody against MT1-MMP (1:100; Santa Cruz Biotechnology, sc-373908 and 1:100; Millipore, MAB3328), neurofilament NF200 (1:500; Sigma, N4142), and synaptophysin (1:1000; Abcam, ab32127) for 2 days at 4°C.

Techniques: Functional Assay

Journal: eLife

Article Title: Site-directed MT1-MMP trafficking and surface insertion regulate AChR clustering and remodeling at developing NMJs

doi: 10.7554/eLife.54379

Figure Lengend Snippet:

Article Snippet: After blocking with 2% BSA and 0.2% Triton X-100 at room temperature for 2 hr, the sections were labeled with primary antibody against MT1-MMP (1:100; Santa Cruz Biotechnology, sc-373908 and 1:100; Millipore, MAB3328), neurofilament NF200 (1:500; Sigma, N4142), and synaptophysin (1:1000; Abcam, ab32127) for 2 days at 4°C.

Techniques: Recombinant, Sequencing, Cell Attachment Assay, Software, Microscopy, Imaging, Electron Microscopy

Journal: Cancers

Article Title: Upregulation of CD36, a Fatty Acid Translocase, Promotes Colorectal Cancer Metastasis by Increasing MMP28 and Decreasing E-Cadherin Expression

doi: 10.3390/cancers14010252

Figure Lengend Snippet: CD36 regulates expression of MMP28. ( A ) Representative gene set enrichment analysis (GSEA) plots generated from RNA-Seq expression data of the HT29 p-Lenti Control and HT29 p-Lenti CD36 overexpression cell lines. The top 10 enriched pathway sets are provided in . Bar codes indicate the location of gene set members in the ranked list of all genes. ES, enrichment score; NES, normalized enrichment score; NOM, nominal p -value; FDR, false discovery adjusted p -value. ( B ) Volcano plot of the HT29 p-Lenti Control and HT29 p-Lenti CD36 overexpression cell lines, showing increased levels of CD36 mRNA associated with an increase in MMP28 mRNA expression. ( C ) qRT-PCR analysis of CD36 and MMP28 mRNA in the HT29, p-Lenti Control and p-Lenti CD36 overexpression cell lines, and in the HT29 LuM3 GFP-Luciferase NTC and shCD36 cell lines. ( D ) qRT-PCR analysis of CD36 and MMP28 mRNA in the HCT116, p-Lenti Control and p-Lenti CD36 overexpression, and HCT116 NTC and CD36 shRNA cell lines. ( E ) Western blot analysis of the HCT116 and HT29 LuM0 p-Lenti Control and p-Lenti CD36 overexpression cell lines for CD36, MMP28, p-Akt and total Akt. ( F ) Western blot analysis of CD36 and MMP28 in HT29 LuM0 and HT29 LuM3 GFP-Luciferase cell lines. ( G ) Confocal microscopy of the HT29 LuM0 and HT29 LuM3 GFP-Luciferase cell lines for actin, CD36 and MMP28. * p < 0.05, ** p < 0.01, *** p < 0.001; SEM.

Article Snippet: Total RNA was isolated using an RNeasy mini kit (QIAGEN). cDNA was synthesized using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Bedford, MA, USA; #4368814). qRT-PCR was carried out using a TaqMan Gene Expression Master Mix (Applied Biosystems, Bedford, MA, USA; #4369016) according to manufacturer’s protocol and TaqMan probes for human CD36 (#4331182-Hs00169627_m1), MMP28 (#4331182-Hs00425232_g1), E-cadherin/CDH1 (#4331182-Hs01023895_m1) and GAPDH (#4331182-Hs02786624_g1) from Thermo Fisher, Waltham, MA, USA.

Techniques: Expressing, Generated, RNA Sequencing, Control, Over Expression, Quantitative RT-PCR, Luciferase, shRNA, Western Blot, Confocal Microscopy

Journal: Cancers

Article Title: Upregulation of CD36, a Fatty Acid Translocase, Promotes Colorectal Cancer Metastasis by Increasing MMP28 and Decreasing E-Cadherin Expression

doi: 10.3390/cancers14010252

Figure Lengend Snippet: MMP28 reduces CRC cell invasion and decreases expression of functional E-cadherin in vitro. ( A ) Fold change in invaded cells/field, absorbance at 560 nm of the de-stained Matrigel trans-well invasion chambers and raw images of the Matrigel trans-well invasion chambers of the HCT116, siControl and siMMP28 cell lines ( n = 3). ( B ) Western blot analysis of the HT29 LuM3 NTC and shCD36 cell lines for CD36, MMP28 and E-cadherin. The long exposure of the same Western blot for E-cadherin is also shown. ( C ) Western blot for MMP28 and E-cadherin expression in HT29LM3 and HT29LuM3 cells. Western blot including the E-cadherin cleavage products CTF1 and CTF2 is shown on a separate blot (long exposure). ( D ) qRT-PCR analysis of the control and MMP28 siRNA transfected HCT116 cell lines for MMP28 and E-cadherin. ( E ) Western blot for MMP28 and E-cadherin expression in HCT116 cells. Western blot including the E-cadherin cleavage products CTF1 and CTF2 is shown on separate blots (long and short exposure). ( F ) Western blot analysis showing the effect of CD36 blocking antibody on E-cadherin expression in HT29 LuM0, HT29LuM3 and HCT116 cells. Expression of E-cadherin is quantified based on band intensity and normalized to actin. ( G ) Western blot analysis showing the effect of CD36 blocking antibody on e-cadherin and MMP28 expression in HCT116 cells treated with IgA or IgCD39 for 48 h in normal medium or SFM (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Total RNA was isolated using an RNeasy mini kit (QIAGEN). cDNA was synthesized using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Bedford, MA, USA; #4368814). qRT-PCR was carried out using a TaqMan Gene Expression Master Mix (Applied Biosystems, Bedford, MA, USA; #4369016) according to manufacturer’s protocol and TaqMan probes for human CD36 (#4331182-Hs00169627_m1), MMP28 (#4331182-Hs00425232_g1), E-cadherin/CDH1 (#4331182-Hs01023895_m1) and GAPDH (#4331182-Hs02786624_g1) from Thermo Fisher, Waltham, MA, USA.

Techniques: Expressing, Functional Assay, In Vitro, Staining, Western Blot, Quantitative RT-PCR, Control, Transfection, Blocking Assay

Journal: Cancers

Article Title: Upregulation of CD36, a Fatty Acid Translocase, Promotes Colorectal Cancer Metastasis by Increasing MMP28 and Decreasing E-Cadherin Expression

doi: 10.3390/cancers14010252

Figure Lengend Snippet: Overexpression of CD36 is associated with an increase in MMP28 expression and reduction in the level of E-cadherin in vivo and human CRC specimens. ( A ) IHC analysis of the tissues from tail-vein injection of the HT29 LuM3 GFP-Luciferase NTC and shCD36 cell lines shown in A. ( B ) Western blot analysis and ( C ) IHC analysis of the tissues from Pt2402 PDX, CD36 low and CD36 high, the isogenic tumors for CD36, MM28 and E-cadherin. ( D ) Western blot analysis of matched normal (N), primary tumor (PT) and liver metastasis (LM) tissues for CD36, MMP28, E-cadherin and CTF2. ( E ) Western blot analysis of CD36, MMP28 and E-cadherin in unmatched N, PT and LM patient tissues.

Article Snippet: Total RNA was isolated using an RNeasy mini kit (QIAGEN). cDNA was synthesized using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Bedford, MA, USA; #4368814). qRT-PCR was carried out using a TaqMan Gene Expression Master Mix (Applied Biosystems, Bedford, MA, USA; #4369016) according to manufacturer’s protocol and TaqMan probes for human CD36 (#4331182-Hs00169627_m1), MMP28 (#4331182-Hs00425232_g1), E-cadherin/CDH1 (#4331182-Hs01023895_m1) and GAPDH (#4331182-Hs02786624_g1) from Thermo Fisher, Waltham, MA, USA.

Techniques: Over Expression, Expressing, In Vivo, Injection, Luciferase, Western Blot

Journal: Journal of Hematology & Oncology

Article Title: The R-RAS2 GTPase is a signaling hub in triple-negative breast cancer cell metabolism and metastatic behavior

doi: 10.1186/s13045-025-01693-3

Figure Lengend Snippet: R-RAS2 interacts with plasma membrane receptors known to be important for breast cancer. a , Selected list of plasma membrane proteins that interact with R-RAS2 in a murine breast cancer tumor isolated from a Rosa26- RRAS2 fl/fl x Wap-Cre female mouse. Full data can be found in Extended Data Table . The known involvement of these proteins in cancer and breast cancer is briefly summarized, and the Mascot Score for the specific affinity column with anti-hemagglutinin (for hemagglutinin-tagged R-RAS2) and a control isotypic immunoglobulin column is also shown. Protein function and association with cancer data have been retrieved from The Human Protein Atlas: https://www.proteinatlas.org . b , Biological processes and pathways found to be significantly altered after KEGG analysis of the R-RAS2 plasma membrane interactome of Extended Data Table . The x-axis shows the percentage or proteins in the pathway found associated to R-RAS2 and the y-axis the–Log10 of the P adjusted value. Full data is provided in Extended Data Table . c , STRING network of physical and functional interactions among the indicated proteins from Fig. 3a. Pink lines indicate experimentally-determined interactions; blue lines indicated interactions found in curated databases; green lines indicate interactions found by textmining and black lines indicate co-expression. d , Co-immunoprecipitation of CD44, Epha2 and Slc3a2 (CD98hc) with R-RRAS2 (IP anti-Hag) was studied in detergent lysates of the CBM-MBC21 mouse cell line. The membranes were re-probed with anti-Hag as a control for loading of R-RAS2, and the arrows indicate the positions of the co-immunoprecipitated proteins and those in the whole cell lysate (WCL). The molecular weight markers are shown on the left. e , Co-immunoprecipitation of CD44, Epha2 and Slc3a2 (CD98hc) with R-RRAS2 in detergent lysates of the human breast cancer cell line BT-549 transfected with a Hag-tagged R-RAS2 construct. Legend as in panel d . f , Mid-plane confocal microscopy sections of CBM-MBC21 cells fixed and stained with R-RAS2 (anti-Hag) and anti-CD44 antibodies to show their co-localization at the plasma membrane. The nucleus of the cells is stained with DAPI (blue). Details of 4 areas of the plasma membranes are shown to the right to show the co-localization of R-RAS2 with CD44 in cell protrusions. g , Co-localization of R-RAS2 and CD44 was measured by analysis of all pixels in 23 cell protusions as in the inset of Fig. 3f and calculating the Pearson’s correlation coefficient. The violin plot shows all data points, the median (= 0.70) and the 75% and 25% percentiles. h , Mid-plane confocal microscopy sections of CBM-MBC21 cells fixed and stained with R-RAS2 (anti-Hag) and anti-CD98hc antibodies to show their co-localization at the plasma membrane. The nucleus of the cells is stained with DAPI (blue). Details of 4 areas of the plasma membranes are shown to the right to show the co-localization of R-RAS2 with CD98hc at the external (apical) plasma membrane of the cell aggregate. i , Co-localization of R-RAS2 and CD98hc was measured by analysis of all pixels in 25 membrane regions as in the inset of Fig. 3h and calculating the Pearson’s correlation coefficient. The violin plot shows all data points, the median (= 0.69) and the 75% and 25% percentiles

Article Snippet: Following blocking, cells were incubated overnight at 4 °C with a mouse monoclonal antibody against R-RAS2 (targeting the Hag tag, 12CA5 clone, Sigma) and rabbit monoclonal antibodies against CD44 or CD98 (Boster Bio and ThermoFisher, respectively).

Techniques: Clinical Proteomics, Membrane, Isolation, Affinity Column, Control, Functional Assay, Expressing, Immunoprecipitation, Molecular Weight, Transfection, Construct, Confocal Microscopy, Staining

Journal: Journal of Hematology & Oncology

Article Title: The R-RAS2 GTPase is a signaling hub in triple-negative breast cancer cell metabolism and metastatic behavior

doi: 10.1186/s13045-025-01693-3

Figure Lengend Snippet: R-RAS2 controls PI3K/Akt, ERK/MAPK and mTORC1 pathway activation in murine RRAS2-overexpressing breast cancer cells. a , Cartoon illustrating RAS/ERK/MAPK, PI3K/Akt and mTORC1 pathway activation by Receptor Tyrosine Kinases (RTKs) like EphaA2, EGFR or HER2, and by amino acid transporters like the CD98hc/CD98lc(LAT1) complex, and by CD44. The positions of some relevant phosphorylated residues are indicated, as well as the putative position of R-RAS2 downstream of the RTKs, CD44 and CD98. b , Western blot analysis of signaling pathway activity based on the phosphorylation of key residues in the elements indicated. Post-nuclear cell lysates of wild type CBM-MBC21 cells and a shRNA-generated RRAS2 knockdown of that cell line were analyzed in the blots. c , Summary of the results generated by Western blot. The inhibitory effect of R-RAS2 depletion is indicated by the number of arrows pointing downwards

Article Snippet: Following blocking, cells were incubated overnight at 4 °C with a mouse monoclonal antibody against R-RAS2 (targeting the Hag tag, 12CA5 clone, Sigma) and rabbit monoclonal antibodies against CD44 or CD98 (Boster Bio and ThermoFisher, respectively).

Techniques: Activation Assay, Western Blot, Activity Assay, Phospho-proteomics, shRNA, Generated, Knockdown

Journal: Journal of Hematology & Oncology

Article Title: The R-RAS2 GTPase is a signaling hub in triple-negative breast cancer cell metabolism and metastatic behavior

doi: 10.1186/s13045-025-01693-3

Figure Lengend Snippet: R-RAS2 co-localizes with CD44 in F-actin-rich cell protrusions of breast cancer cells. a , Confocal microscopy sections at the plane of contact with the coverslips of CBM-MBC21 control and knockdown cells plated on coverslips coated with poly-L-lysine alone or with poly-L-lysine plus hyaluronic acid (HA). Cells were plated in the presence or absence of a blocking anti-CD44 antibody. After incubation, cells were fixed and stained with R-RAS2 (anti-Hag) and phalloidin to show their co-localization at sites rich in F-actin. The nucleus of the cells is stained with DAPI (blue). b , Co-localization of R-RAS2 and F-actin at cell protrusions was measured by analysis of all pixels in 17–33 cell protrusions and calculating the Pearson’s correlation coefficient. The violin plot shows all data points, the median and the 75% and 25% percentiles for each condition. Statistical significance was assessed using a one-way ANOVA Tukey’s multiple comparison test. ***, p < 0.001, ****, p < 0.0001. c , Cell area at the contact site with the coverslip in the experiment of Fig. 6a-b, was calculated for 7–28 cells per condition. The violin plot shows all data points, the median and the 75% and 25% percentiles for each condition. Statistical significance was assessed using a one-way ANOVA Tukey’s multiple comparison test. *, p = 0.03; **, p = 0.008; ***, p = 0.0003; ****, p < 0.0001. d , The number and distance to the center of cell protrusions were measured in phalloidin stains by calculating the center of mass and drawing lines (yellow) from the center to the tip of cell protrusions. e , The number of protrusions was calculated as in Fig. 6d for a minimum of 15 cells per culture condition. The violin plot shows all data points, the median and the 75% and 25% percentiles for each condition. Statistical significance was assessed using a one-way ANOVA Tukey’s multiple comparison test. *, p < 0.05; **, p = 0.002; ****, p < 0.0001. f , The distance to the cell center of mass of protrusions was calculated as in Fig. 6d for a minimum of 100 cell protrusions per culture condition. The violin plot shows all data points, the median and the 75% and 25% percentiles for each condition. Statistical significance was assessed using a one-way ANOVA Tukey’s multiple comparison test. *, p < 0.05; ****, p < 0.0001

Article Snippet: Following blocking, cells were incubated overnight at 4 °C with a mouse monoclonal antibody against R-RAS2 (targeting the Hag tag, 12CA5 clone, Sigma) and rabbit monoclonal antibodies against CD44 or CD98 (Boster Bio and ThermoFisher, respectively).

Techniques: Confocal Microscopy, Control, Knockdown, Blocking Assay, Incubation, Staining, Comparison

Journal: Journal of Hematology & Oncology

Article Title: The R-RAS2 GTPase is a signaling hub in triple-negative breast cancer cell metabolism and metastatic behavior

doi: 10.1186/s13045-025-01693-3

Figure Lengend Snippet: R-RAS2 is downstream of CD44 promoting migratory, invasive and metastatic behavior of breast cancer cells. a , Box and whiskers plot showing all experimental points of a migration assay in Boyden chambers separated by a 8 μm-diameter pore membrane. Cells were incubated overnight (~16 h). Each point represents the mean of cells per surface unit (mm 2 ) of membrane counted. A number of 18 areas of the membrane were counted per experimental condition. Statistical significance was assessed by carrying out a Mann-Whitney test. b , Box and whiskers plot showing all experimental points of a migration assay in Boyden chambers separated by a 8 μm-diameter pore membrane and layered on the upper chamber with 100 μL of Matrigel. Cells were incubated overnight (~16 h). Each point represents the mean of cells per surface unit (mm 2 ) of membrane counted. A number of 18 areas of the membrane were counted per experimental condition. Statistical significance was assessed by carrying out a Mann-Whitney test. c , Light field microscopy of the bottom part of the membrane with 8-μm diameter pores separating an upper Boyden chamber in which CBM-MBC21 control of knockdown cells were seeded in serum-free medium and a lower chamber containing medium with 20% feta bovine serum. In addition, the upper chamber had a layer or either Matrigel or Matrigel + hyaluronic acid directly on top of the membrane. Cells were allowed to migrate for 8 h from the top to the bottom chambers and through the Matrigel or Matrigel + HA layers. The bottom part of the membrane was stained with crystal violet to count the number of migrated cells. d , Box and whiskers plot showing all experimental points of the experiment illustrated in Fig. 8c. Each point represents the mean of cells per surface unit (mm 2 ) of membrane counted. A number of 14 areas of the membrane were counted per experimental condition. Statistical significance was assessed using unpaired t-tests. e , An invasion assay as this of Fig. 8 b and d, was carried out in Boyden chambers coated in the upper chamber with a layer of Matrigel + hyaluronic acid. Control and knockdown CBM-MBC21 cells were incubated or not (no Ab) with 5 μg/mL of blocking anti-CD44 antibody in the upper chamber for 8 h. Box and whiskers plot showing all experimental points that represent the mean of cells per surface unit (mm 2 ) of membrane counted. A number of 13–14 areas of the membrane were counted per experimental condition. Statistical significance was assessed using a one-way ANOVA Tukey’s multiple comparison test. f , The metastatic capacity of control and knockdown CBM-MBC21 cells 25 days after orthotopic inoculation in the left inguinal mammary gland of female C57BL/6 mice was assessed by flow cytometry of the lungs and liver. The pseudocolor plot shows the identification of metastatic breast cancer cells in the lungs according to the expression of GFP and CD44. g , Box and whiskers plot showing all experimental points calculated as illustrated in Fig. 8f and Suppl Fig. f referring to BC cell infiltration of lungs and liver, respectively. Each point represents a single mouse. Statistical significance was assessed by carrying out Mann-Whitney tests. h , Box and whiskers plot showing all experimental points indicating primary tumor weight in mice inoculated with BT-549 human BC cells at the day of sacrifice (day 39). Statistical significance was assessed by carrying out a Mann-Whitney test. i , Photograph of the entire liver taken from a mouse inoculated with wild type BT-549, control, BC cells. The blue arrow points at the presence of a green fluorescent BC tumor nodule. j , The metastatic capacity of control and knockdown BT-549 cells 39 days after orthotopic inoculation in the left inguinal mammary gland of female Rag2 −/− γc −/− mice was assessed by flow cytometry of the lungs and liver. The two-color plot shows the identification of metastatic breast cancer cells in the lungs according to the expression of GFP and human EGFR. k , Box and whiskers plot showing all experimental points calculated as illustrated in Fig. 8j referring to BC cell infiltration of lungs and liver. One out of four liver lobules per animal were processed for flow cytometry analysis. Each point represents a single mouse. Statistical significance was assessed by carrying out Mann-Whitney tests

Article Snippet: Following blocking, cells were incubated overnight at 4 °C with a mouse monoclonal antibody against R-RAS2 (targeting the Hag tag, 12CA5 clone, Sigma) and rabbit monoclonal antibodies against CD44 or CD98 (Boster Bio and ThermoFisher, respectively).

Techniques: Migration, Membrane, Incubation, MANN-WHITNEY, Microscopy, Control, Knockdown, Staining, Invasion Assay, Blocking Assay, Comparison, Flow Cytometry, Expressing

Journal: Respiratory Research

Article Title: Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) promotes lung fibroblast proliferation, survival and differentiation to myofibroblasts

doi: 10.1186/s12931-016-0334-7

Figure Lengend Snippet: EMMPRIN overexpression in normal human lung fibroblasts (NHLF): NHLF were transiently transfected with either EMMPRIN/GFP or GFP alone and assessed for EMMPRIN expression using ( a ) Fluorescent microscopy demonstrating the distribution of GFP throughout the cell (left panel) in GFP control cells, while EMMPRIN-GFP cells primarily display cell-surface GFP expression (right panel). White Bar = 50 um. b Western blot analysis and ( c ) Gelatin zymography for MMP-2 release and activation (n = 10)

Article Snippet: Primary antibodies included mouse monoclonal anti- α-SMA (1:10000 dilution, Sigma Aldrich), anti-human EMMPRIN (1:1000 dilution, Santa Cruz Biotechnology), rabbit polyclonal anti-Caspase 3 (1:500 dilution, Santa Cruz Biotechnology), rabbit polyclonal anti β-Catenin (1:1000 dilution, Abcam, Cambridge, MA) and β-Tubulin (1:200 dilution; Santa Cruz Biotechnology).

Techniques: Over Expression, Transfection, Expressing, Microscopy, Control, Western Blot, Zymography, Activation Assay

Journal: Respiratory Research

Article Title: Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) promotes lung fibroblast proliferation, survival and differentiation to myofibroblasts

doi: 10.1186/s12931-016-0334-7

Figure Lengend Snippet: EMMPRIN overexpression induces phenotypic changes in normal human lung fibroblasts. Growth arrested NHLF transiently transfected with either EMMPRIN/GFP or GFP were treated with TGF- β1 5 and 10 ng/ml for 48 h. a Cell proliferation was assessed by thymidine incorporation. b Cell migration using Modified Boyden chamber. Each bar represents means ± SEM of 3 independent experiments each run in duplicates. ** p < 0.001 comparing GFP to EMMPRIN/GFP transfected cells, # p < 0.01 Comparing TGF- β1 treated cells to control. c A representative Western blot for α – smooth actin (α-SMA) of cell lysates from EMMPRIN/GFP and GFP control cells in the presence and absence of TGF-β1. β-Tubulin was used as the loading control. Image is representative of 3 independent experiments each run in duplicate

Article Snippet: Primary antibodies included mouse monoclonal anti- α-SMA (1:10000 dilution, Sigma Aldrich), anti-human EMMPRIN (1:1000 dilution, Santa Cruz Biotechnology), rabbit polyclonal anti-Caspase 3 (1:500 dilution, Santa Cruz Biotechnology), rabbit polyclonal anti β-Catenin (1:1000 dilution, Abcam, Cambridge, MA) and β-Tubulin (1:200 dilution; Santa Cruz Biotechnology).

Techniques: Over Expression, Transfection, Migration, Modification, Control, Western Blot

Journal: Respiratory Research

Article Title: Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) promotes lung fibroblast proliferation, survival and differentiation to myofibroblasts

doi: 10.1186/s12931-016-0334-7

Figure Lengend Snippet: EMMPRIN overexpression induces resistance of normal human lung fibroblasts to apoptosis: Growth arrested NHLF overexpressing either EMMPRIN/GFP or GFP were treated with TGF- β1 from 0 to 10 ng/ml for 24 h in the presence and absence of 0.5 mM of Staurosporine (STS) added for 3 h prior to the end of the experiments. Apoptosis was measured by FACS analysis ( a - b ) using annexin V/PI staining and cell death detection ELISA assay ( c ). Relative apoptosis is expressed as a percentage of the assay-positive control that was run on the ELISA plate for each experiment. All samples were run in triplicate for each ELISA and FACS analysis. ** p < 0.001 comparing GFP to EMMPRIN/GFP transfected cells (n = 3 independent experiments)

Article Snippet: Primary antibodies included mouse monoclonal anti- α-SMA (1:10000 dilution, Sigma Aldrich), anti-human EMMPRIN (1:1000 dilution, Santa Cruz Biotechnology), rabbit polyclonal anti-Caspase 3 (1:500 dilution, Santa Cruz Biotechnology), rabbit polyclonal anti β-Catenin (1:1000 dilution, Abcam, Cambridge, MA) and β-Tubulin (1:200 dilution; Santa Cruz Biotechnology).

Techniques: Over Expression, Staining, Enzyme-linked Immunosorbent Assay, Positive Control, Transfection

Journal: Respiratory Research

Article Title: Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) promotes lung fibroblast proliferation, survival and differentiation to myofibroblasts

doi: 10.1186/s12931-016-0334-7

Figure Lengend Snippet: TGF-β1 induces EMMPRIN Expression and MMP-2 activation in normal human lung fibroblasts; inhibition of MMP-2 activation by EMMPRIN functional blocking antibody: ( a ) A representative Western blot showing the expression of EMMPRIN in the cell lysates from NHLF stimulated with TGF-β1 compared to control untreated cells. β-Tubulin was used as the loading control. b A representative zymography demonstrating MMP-2 activation in the conditioned media from NHLF stimulated with TGF-β1 compared to the control untreated cells and inhibition of MMP-2 activation by EMMPRIN functional blocking antibody (n = 3 independent experiments, each run in duplicate)

Article Snippet: Primary antibodies included mouse monoclonal anti- α-SMA (1:10000 dilution, Sigma Aldrich), anti-human EMMPRIN (1:1000 dilution, Santa Cruz Biotechnology), rabbit polyclonal anti-Caspase 3 (1:500 dilution, Santa Cruz Biotechnology), rabbit polyclonal anti β-Catenin (1:1000 dilution, Abcam, Cambridge, MA) and β-Tubulin (1:200 dilution; Santa Cruz Biotechnology).

Techniques: Expressing, Activation Assay, Inhibition, Functional Assay, Blocking Assay, Western Blot, Control, Zymography

Journal: Respiratory Research

Article Title: Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) promotes lung fibroblast proliferation, survival and differentiation to myofibroblasts

doi: 10.1186/s12931-016-0334-7

Figure Lengend Snippet: EMMPRIN functional blocking antibody induces apoptosis in normal human lung fibroblasts: NHLF treated for 24 h with TGF- β1 in the presence of either EMMPRIN functional blocking antibody or IgG control antibody. Apoptosis was measured by ( a ) FACS analysis using annexin V/PI staining; ( b ) Cell Death Detection ELISA assay, each bar represents means ± SEM of 3 independent experiments, each run in duplicate, ** p < 0.001 comparing EMMPRIN functional blocking antibody to IgG control antibody treated cells; and ( c ) Western blot for caspase- 3 of cell lysates from NHLF treated with TGF-β1 (5 and 10 ng/ml) in the presence of EMMPRIN functional blocking antibody or IgG control antibody. β-Tubulin was used as the loading control. Image is a representative of 3 independent experiments, each run in duplicate

Article Snippet: Primary antibodies included mouse monoclonal anti- α-SMA (1:10000 dilution, Sigma Aldrich), anti-human EMMPRIN (1:1000 dilution, Santa Cruz Biotechnology), rabbit polyclonal anti-Caspase 3 (1:500 dilution, Santa Cruz Biotechnology), rabbit polyclonal anti β-Catenin (1:1000 dilution, Abcam, Cambridge, MA) and β-Tubulin (1:200 dilution; Santa Cruz Biotechnology).

Techniques: Functional Assay, Blocking Assay, Control, Staining, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Respiratory Research

Article Title: Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) promotes lung fibroblast proliferation, survival and differentiation to myofibroblasts

doi: 10.1186/s12931-016-0334-7

Figure Lengend Snippet: EMMPRIN blocking antibody attenuates TGF-β1 induced lung fibroblast proliferation, migration and differentiation to myofibroblast: NHLF were stimulated with TGF-β1 (0-10 ng/ml) in the presence of either EMMPRIN functional blocking antibody or IgG control antibody examined for ( a ) Cells proliferation using [3H] thymidine incorporation; ( b ) Cell migration using a modified Boyden chamber assay. Each bar represents means ± SEM of 3 independent experiments, each run in duplicate, # p = 0.002 or ** p < 0.001 comparing EMMPRIN functional blocking antibody to IgG control antibody treated cells; and ( c ) Cell differentiation using Western blot for α-SMA of cell lysates from NHLF treated with TGF-β1 (5 and 10 ng/ml) in the presence of either EMMPRIN functional blocking antibody or IgG antibody. β-Tubulin was used as the loading control. Image is a representative of 3 independent experiments, each run in duplicate

Article Snippet: Primary antibodies included mouse monoclonal anti- α-SMA (1:10000 dilution, Sigma Aldrich), anti-human EMMPRIN (1:1000 dilution, Santa Cruz Biotechnology), rabbit polyclonal anti-Caspase 3 (1:500 dilution, Santa Cruz Biotechnology), rabbit polyclonal anti β-Catenin (1:1000 dilution, Abcam, Cambridge, MA) and β-Tubulin (1:200 dilution; Santa Cruz Biotechnology).

Techniques: Blocking Assay, Migration, Functional Assay, Control, Modification, Boyden Chamber Assay, Cell Differentiation, Western Blot

Journal: Respiratory Research

Article Title: Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) promotes lung fibroblast proliferation, survival and differentiation to myofibroblasts

doi: 10.1186/s12931-016-0334-7

Figure Lengend Snippet: EMMPRIN Overexpression Increases Wnt/β-catenin signaling: ( a ) Representative Western blot for phosphorylated β-catenin of cells lysates from NHLF overexpressing either EMMPRIN/GFP or GFP treated with and without TGF-β1 (10 ng/ml). β-Tubulin was used as the loading control. Image is representative of 3 independent experiments, each run in duplicate. b Transient transfection of TOPFLASH, FOPFLASH, and control renilla luciferase reporter constructs into NHLF overexpressing either EMMPRIN/GFP or GFP then treated with TGF-β1 10 ng/ml. Each bar represents means ± SEM of 3 independent experiments, each run in duplicate, ** p < 0.001comparing GFP to EMMPRIN/GFP transfected cells

Article Snippet: Primary antibodies included mouse monoclonal anti- α-SMA (1:10000 dilution, Sigma Aldrich), anti-human EMMPRIN (1:1000 dilution, Santa Cruz Biotechnology), rabbit polyclonal anti-Caspase 3 (1:500 dilution, Santa Cruz Biotechnology), rabbit polyclonal anti β-Catenin (1:1000 dilution, Abcam, Cambridge, MA) and β-Tubulin (1:200 dilution; Santa Cruz Biotechnology).

Techniques: Over Expression, Western Blot, Control, Transfection, Luciferase, Construct