Journal: Molecular cell

Article Title: KAP1 is a Chromatin Reader that Couples Steps of RNA Polymerase II Transcription to Sustain Oncogenic Programs

doi: 10.1016/j.molcel.2020.04.024

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: hFAB Rhodamine anti-Actin , Bio-Rad , Cat# 12004166.

Techniques: Virus, Mutagenesis, Recombinant, Membrane, Protease Inhibitor, SYBR Green Assay, Western Blot, Staining, Plasmid Preparation, RNA HS Assay, Reverse Transcription, Bicinchoninic Acid Protein Assay, Cloning, shRNA, Software

Journal: Cell reports

Article Title: Development of single-molecule ubiquitination mediated fluorescence complementation to visualize protein ubiquitination dynamics in dendrites

doi: 10.1016/j.celrep.2022.111658

Figure Lengend Snippet:

Article Snippet: Anti-GAPDH hFAB TM rhodamine antibody, human FAB, one-step detection , BioRad , BioRad, #12004167.

Techniques: Virus, Recombinant, Fluorescence, In Situ, Subcloning, Plasmid Preparation, Mutagenesis, Software, Transfection, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Magnetic Beads, Western Blot

Journal: Cell Death & Disease

Article Title: The deubiquitinase USP9X and E3 ligase WWP1 orchestrate IGF2BP2 ubiquitination homeostasis to drive TNBC progression and cisplatin sensitivity

doi: 10.1038/s41419-025-08038-5

Figure Lengend Snippet: a Demonstration of the PTMs locus of IGF2BP2 by the PhosphoSitePlus database. b Flowchart showing the route to construct the siRNA screen; IGF2BP2 expression was assessed by Western blot upon DUB knockdown, with densitometric analysis of band gray values. c A positive correlation was observed between IGF2BP2 and USP9X protein levels in Pan-Cancer Proteome Atlas (TPCPA)-BRCA TNBC ( R = 0.28, P = 0.031), with missing values imputed using a k-nearest neighbors- based algorithm. d IGF2BP2 activity is higher in USP9X-high tumors than in USP9X-low tumors in the FUSCC TNBC cohort. e , f Expression levels of USP9X in MDA-MB-231 and Hs 578T cells transfected with knockdown USP9X (shUSP9X) and blank control (shRNA-NC) were detected by qRT-PCR and Western blot. g , h Western blot analysis of IGF2BP2 protein levels in MDA-MB-231 and Hs 578T cells stably transfected with shUSP9X or control shRNA (shRNA-NC) following treatment with 50 μg/ml cycloheximide (CHX) for the indicated time points (0, 2, 4, 6, 8 h). Quantification of grayscale values from all experimental groups was performed to determine the protein degradation half-life (t₁/₂) of IGF2BP2. i , j HEK 293T cells were co-transfected with HIS-IGF2BP2 and FLAG-USP9X, followed by treatment with 50 μg/ml CHX to inhibit protein synthesis. Cells were harvested at the indicated time points (0, 2, 4, 6, 8 h), and protein levels were analyzed by Western blot using anti-HIS (for IGF2BP2) and anti-FLAG (for USP9X) antibodies. Quantification of grayscale values from all experimental groups was performed to determine the protein degradation half-life (t₁/₂) of HIS-IGF2BP2. Error bars indicate mean ( n = 3) ± standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Further, we purified the HIS-tagged IGF2BP2 full length protein and performed in vitro protein interaction pull-down experiments with the purified protein of the GST-tagged USP9X M3 segment (HY-P701431, MCE, NJ, USA) to further clarify its interconnections (Fig. ).

Techniques: Construct, Expressing, Western Blot, Knockdown, Activity Assay, Transfection, Control, shRNA, Quantitative RT-PCR, Stable Transfection, Standard Deviation

Journal: Cell Death & Disease

Article Title: The deubiquitinase USP9X and E3 ligase WWP1 orchestrate IGF2BP2 ubiquitination homeostasis to drive TNBC progression and cisplatin sensitivity

doi: 10.1038/s41419-025-08038-5

Figure Lengend Snippet: a Cell lysates of MDA-MB-231 and Hs 578T were pulled down by IP using antibodies against USP9X and IGF2BP2, followed by Western blot detection. b , c Confocal images showing co-localization of USP9X (red) and IGF2BP2 (green) in MDA-MB-231 and Hs 578T cells, and nuclei were restained with DAPI (blue), followed by quantitative analysis of fluorescence intensity. d Domain organization schematics of FLAG-USP9X, HIS-IGF2BP2 and their engineered truncation constructs, depicting functional domains and truncation boundaries. e HEK 293T cells were co-transfected with HIS-IGF2BP2 and FLAG-tagged USP9X full-length or truncated mutants. Cell lysates were analyzed by IP with FLAG beads and then detected by Western blot with antibodies against HIS and FLAG. f HEK 293T cells were cotransfected with FLAG-USP9X and HIS-tagged full-length or truncated mutants of IGF2BP2. Cell lysates were analyzed by IP with HIS beads and then detected by Western blot with antibodies against FLAG and HIS. g HDock ( http://hdock.phys.hust.edu.cn/ ) analysis showing details of the interaction between USP9X-M3 and IGF2BP2-M3, the protein models were retrieved and downloaded at UniProt ( https://www.uniprot.org/ ). h Purified GST-USP9X-M3 and HIS-IGF2BP2 were incubated in binding buffer at 4 °C for 2 h, Complexes were pulled down with HIS/GST magnetic beads, Input (10%) and bound fractions were analyzed by Western blot.

Article Snippet: Further, we purified the HIS-tagged IGF2BP2 full length protein and performed in vitro protein interaction pull-down experiments with the purified protein of the GST-tagged USP9X M3 segment (HY-P701431, MCE, NJ, USA) to further clarify its interconnections (Fig. ).

Techniques: Western Blot, Fluorescence, Construct, Functional Assay, Transfection, Purification, Incubation, Binding Assay, Magnetic Beads

Journal: Cell Death & Disease

Article Title: The deubiquitinase USP9X and E3 ligase WWP1 orchestrate IGF2BP2 ubiquitination homeostasis to drive TNBC progression and cisplatin sensitivity

doi: 10.1038/s41419-025-08038-5

Figure Lengend Snippet: a , b Total proteins were extracted from control and USP9X-knockdown MDA-MB-231 and Hs 578T cells treated with DMSO, MG132, or CQ for 8 h. IGF2BP2 protein levels were analyzed by Western blot, and band intensities were quantified for statistical comparison. Error bars indicate mean ( n = 3) ± standard deviation. c , d MDA-MB-231 and Hs 578T cells were co-transfected with HA-Ub along with either control siRNA, USP9X-targeting siRNA (siUSP9X), or FLAG-USP9X plasmid. Following MG-132 (20 μM, 6 h) treatment to stabilize ubiquitinated proteins, IGF2BP2 was immunoprecipitated and its ubiquitination status was analyzed by Western blot using anti-HA antibody. e , f HEK 293T cells were cotransfected using HIS-tagged IGF2BP2 with HA-Ub, Lys48 only, Lys63 only, Lys48R, Lys63R plasmids, respectively, and after treatment with 20 μM MG-132 for 6 h. HIS pull-down assays were performed and ubiquitination patterns were analyzed by immunoblotting with anti-HA for ubiquitin conjugates and anti-HIS for IGF2BP2 loading control. g HA-Ub wild-type or Ub-Lys48R was transfected in MDA-MB-231 and Hs 578T cells in control or knockdown USP9X groups. After 72 h, total protein was extracted and IGF2BP2 levels were analyzed by Western blot using anti-IGF2BP2 antibody, with USP9X detection serving as knockdown verification. h HEK293T cells were co-transfected with FLAG-USP9X, HA-Ub, and either wild-type HIS-IGF2BP2 or its lysine-to-arginine (K-R) mutants. Following MG-132 treatment (20 μM, 6 h) to stabilize ubiquitinated proteins, HIS-tagged IGF2BP2 was immunoprecipitated and ubiquitination patterns were analyzed by immunoblotting with anti-HA (ubiquitin), anti-HIS (IGF2BP2), and anti-FLAG (USP9X). i HEK 293T cells were cultured for 72 h after co-transfection with empty or siUSP9X using wild-type Ub, HIS-IGF2BP2 or HIS-IGF2BP2487/583R plasmids, and total proteins were collected and subjected to Western blot. Quantification of HIS-IGF2BP2 protein levels normalized to β-Actin. j HIS-tagged IGF2BP2-WT or HIS-IGF2BP2-487/583R was used to co-transfect with HA-Ub and FLAG-USP9X, and ubiquitin ligation of IGF2BP2 was analyzed after collection of total proteins after treatment with 20 μM MG-132 for 6 h and IP pull-down using HIS antibody.

Article Snippet: Further, we purified the HIS-tagged IGF2BP2 full length protein and performed in vitro protein interaction pull-down experiments with the purified protein of the GST-tagged USP9X M3 segment (HY-P701431, MCE, NJ, USA) to further clarify its interconnections (Fig. ).

Techniques: Control, Knockdown, Western Blot, Comparison, Standard Deviation, Transfection, Plasmid Preparation, Immunoprecipitation, Ubiquitin Proteomics, Cell Culture, Cotransfection, Ligation

Journal: Cell Death & Disease

Article Title: The deubiquitinase USP9X and E3 ligase WWP1 orchestrate IGF2BP2 ubiquitination homeostasis to drive TNBC progression and cisplatin sensitivity

doi: 10.1038/s41419-025-08038-5

Figure Lengend Snippet: a Cell lysates of MDA-MB-231 and Hs 578T were pulled down by IP using antibodies against WWP1 and IGF2BP2, followed by Western blot detection. b HEK293T cells were co-transfected with HIS-tagged IGF2BP2 and FLAG-tagged WWP1, treated with CHX (50 μg/ml) and total proteins were collected at the corresponding time and then detected by Western blot, and the gray values were calculated for statistical analysis. Error bars indicate mean ( n = 3) ± standard deviation. c – f MDA-MB-231 and Hs 578T were co-transfected with HA-Ub using the indicated controls, siWWP1 or FLAG-tagged WWP1, and after treatment with 20 μM MG-132 for 6 h. IGF2BP2 was immunoprecipitated and its ubiquitination status was analyzed by immunoblotting with anti-HA antibody (for ubiquitin conjugates) and anti-IGF2BP2 antibody (for protein input control). g HA-Ub wild-type or Ub-Lys48R was transfected in MDA-MB-231 and Hs 578T cells transfected with empty vector or FLAG-tagged WWP1 groups, and the total proteome was collected after 72 h and detected by Western blot using IGF2BP2 and FLAG antibodies. h HEK 293T cells were cultured with wild-type Ub, HIS-IGF2BP2 or HIS-IGF2BP2-487/583R plasmids co-transfected with null or FLAG-WWP1 for 72 h. Total proteins were collected and subjected to Western blot. i HEK293T cells were co-transfected with HIS-IGF2BP2 and empty vector (control), FLAG-WWP1, FLAG-USP9X, or both FLAG-WWP1 and FLAG-USP9X, along with wild-type ubiquitin. After 72 h, total protein was extracted and IGF2BP2 expression levels were analyzed by immunoblotting with anti-HIS antibody, while ectopic WWP1 and USP9X expression was verified using anti-FLAG antibody. j HEK 293T cells were cotransfected with HA-Ub, HIS-IGF2BP2 with empty or FLAG-USP9X or FLAG-USP9X and FLAGWWP1 and treated with 20 μM MG-132 for 6 h. After treatment with HIS antibody, the cells were subjected to IP pull-down and the ubiquitination levels of HIS-IGF2BP2 were ultimately assessed by Western blot analysis.

Article Snippet: Further, we purified the HIS-tagged IGF2BP2 full length protein and performed in vitro protein interaction pull-down experiments with the purified protein of the GST-tagged USP9X M3 segment (HY-P701431, MCE, NJ, USA) to further clarify its interconnections (Fig. ).

Techniques: Western Blot, Transfection, Standard Deviation, Immunoprecipitation, Ubiquitin Proteomics, Control, Plasmid Preparation, Cell Culture, Expressing

Journal: Cell Death & Disease

Article Title: The deubiquitinase USP9X and E3 ligase WWP1 orchestrate IGF2BP2 ubiquitination homeostasis to drive TNBC progression and cisplatin sensitivity

doi: 10.1038/s41419-025-08038-5

Figure Lengend Snippet: a CCK-8 assay was used to examine the IC 50 value of Cisplatin (CDDP, MCE, HY-17394). b Scatter plot of IGF2BP2 and USP9X expression in individual patients, with color indicating cisplatin response status. Dotted lines represent median values of IGF2BP2 and USP9X expression, used to define high and low expression subgroups. Fisher ’s exact test was used to compare the IGF2BP2High/USP9XHigh group against each of the other three subgroups. The response rate was significantly higher than that in the IGF2BP2Low/USP9XHigh group ( P = 0.003) and the IGF2BP2Low/USP9XLow group ( P = 0.020), and showed a non-significant trend toward higher response compared to the IGF2BP2High/USP9XLow group ( P = 0.179). Heatmap colors indicate response rates. c , d . Expression levels of USP9X, WWP1 and IGF2BP2 in MDA-MB-231 and Hs 578T cells from saline control (NC) and cisplatin drug-treated (CDDP) groups were detected by qRT-PCR and Western blot. e HA-Ub was transfected in NC and CDDP group cells, respectively, and IP pull-down was performed using IGF2BP2 antibody and detected by Western blot. f , g Confocal images showing co-localization of USP9X or WWP1 (red) and IGF2BP2 (green) in MDA-MB-231 and Hs 578T cells after 4 h treatment with saline or CDDP for 4 h, with nuclei were restained with DAPI (blue). h His-tagged IGF2BP2-M3 was overexpressed in IGF2BP2-knockdown MDA-MB-231 and Hs 578T cells treated with cisplatin or saline control. Total proteins were IP using anti-HIS antibody, followed by Western Blot assays with antibodies against USP9X, WWP1, and HIS-tag. i Biotin-labeled cisplatin probes were synthesized for pull-down assay and the enriched proteins were used for mass spectrometry identification. The identification results comprehensively listed all unique peptide sequences specific to USP9X. j Cell lysates from MDA-MB-231, Hs 578T, and HEK293T cells were incubated with biotin-labeled cisplatin probes or biotin-only controls coupled to streptavidin magnetic beads. After pull-down and denaturation, captured proteins were analyzed by Western blot with USP9X antibody to specifically detect cisplatin-USP9X interactions. k Four FLAG-tagged USP9X truncation mutants were purified as recombinant proteins for in vitro binding assays. These proteins were incubated with streptavidin magnetic beads conjugated to either biotin-cisplatin or biotin alone. Bound proteins were eluted and analyzed by immunoblotting with anti-FLAG antibody. Error bars indicate mean ( n = 3) ± standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Further, we purified the HIS-tagged IGF2BP2 full length protein and performed in vitro protein interaction pull-down experiments with the purified protein of the GST-tagged USP9X M3 segment (HY-P701431, MCE, NJ, USA) to further clarify its interconnections (Fig. ).

Techniques: CCK-8 Assay, Expressing, Saline, Control, Quantitative RT-PCR, Western Blot, Transfection, Knockdown, Labeling, Synthesized, Pull Down Assay, Mass Spectrometry, Incubation, Magnetic Beads, Purification, Recombinant, In Vitro, Binding Assay, Standard Deviation

Journal: Cell Death & Disease

Article Title: The deubiquitinase USP9X and E3 ligase WWP1 orchestrate IGF2BP2 ubiquitination homeostasis to drive TNBC progression and cisplatin sensitivity

doi: 10.1038/s41419-025-08038-5

Figure Lengend Snippet: a Working model of IGF2BP2 targeting MYC and CDK6 mRNAs in an m6A-dependent manner to promote TNBC progression. b – e qRT-PCR was performed to measure the transcript levels of CDK6 and MYC within IGF2BP2 or IgG immunocomplexes in saline or cisplatin-treated MDA-MB-231 and Hs 578T cell lysates. Relative levels of all genes were normalized with IgG. f After treatment with CHX (50 μg/ml), the corresponding total proteins were collected at different time points of cisplatin addition and protein abundance of IGF2BP2 and CDK6 was detected by Western blot. g Protein abundance of IGF2BP2 and CDK6 was made to be converted to gray values and analyzed statistically. h mRNA half-life (t1/2) was measured in cisplatin-treated vs control cells using actinomycin D (5 μg/mL) chase (0, 0.5, 1 h) followed by qRT-PCR. i MDA-MB-231 and Hs 578 T cells were treated with either saline or cisplatin for 4 h. IGF2BP2-containing complexes were immunoprecipitated from cell lysates and analyzed by immunoblotting for interacting partners (HUR and EIF4A1), with IGF2BP2 serving as the immunoprecipitation control. Error bars indicate mean ( n = 3) ± standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Further, we purified the HIS-tagged IGF2BP2 full length protein and performed in vitro protein interaction pull-down experiments with the purified protein of the GST-tagged USP9X M3 segment (HY-P701431, MCE, NJ, USA) to further clarify its interconnections (Fig. ).

Techniques: Quantitative RT-PCR, Saline, Quantitative Proteomics, Western Blot, Control, Immunoprecipitation, Standard Deviation

Journal: Cell Death & Disease

Article Title: The deubiquitinase USP9X and E3 ligase WWP1 orchestrate IGF2BP2 ubiquitination homeostasis to drive TNBC progression and cisplatin sensitivity

doi: 10.1038/s41419-025-08038-5

Figure Lengend Snippet: Mechanistically, USP9X and WWP1 coordinately control IGF2BP2 stability through ubiquitination modulation. Cisplatin (CDDP) binding to USP9X disrupts this balance. Therapeutically, combining WP1130 (USP9X inhibitor) with cisplatin ablates IGF2BP2’s m6A-binding activity toward MYC and CDK6 mRNAs, providing an effective strategy against TNBC progression.

Article Snippet: Further, we purified the HIS-tagged IGF2BP2 full length protein and performed in vitro protein interaction pull-down experiments with the purified protein of the GST-tagged USP9X M3 segment (HY-P701431, MCE, NJ, USA) to further clarify its interconnections (Fig. ).

Techniques: Control, Ubiquitin Proteomics, Binding Assay, Activity Assay

Journal: Oncoimmunology

Article Title: Novel immunotheranostic FAP-inhibitor target modules for imaging and elimination of FAP-positive cells by UniCAR T-cells

doi: 10.1080/2162402X.2025.2598908

Figure Lengend Snippet: Assessment of the binding properties of FAPI TMs to FAP-positive target cells. The surface expression of FAP on HT1080 hFAP (a) and MRC-5 (b) cells was assessed by flow cytometry using commercial anti-hFAP mAb and binding was detected using goat anti-mouse IgG Pacific Blue. Data are shown for one representative experiment out of three independent experiments with similar results ( n = 3). The number of hFAP surface antigens per cell was determined for the HT1080 hFAP and MRC-5 cell lines using QIFIKIT ( n = 1) (c). The FAP-specific binding of 64 Cu-radiolabeled FAPI TMs to HT1080 hFAP cells was evaluated in vitro after 1 h of incubation at 37 °C ( n = 1) (d). The binding of NODA-GA-FAPI-PEG 4/12/24 -E5B9 TMs and NODA-GA-FAPI-PEG 4 to HT1080 hFAP was investigated using flow cytometry. The target cells were incubated with 200 nM of either NODA-GA-FAPI-PEG 4 (black), NODA-GA-FAPI-PEG 4 -E5B9 (red), NODA-GA-FAPI-PEG 12 -E5B9 (blue) or NODA-GA-FAPI-PEG 24 -E5B9 (green), and binding was detected using anti-La mAb 5B9 (e). The data are shown for one representative experiment out of three independent experiments with similar results ( n = 3). The binding affinity of NODA-GA-FAPI-PEG 12 -E5B9 (blue) and NODA-GA-FAPI-PEG 24 -E5B9 (green) TMs to HT1080 hFAP (f, g) and MRC-5 (h) cell lines were further studied by flow cytometry using anti-La mAb 5B9 and the goat anti-mouse IgG Pacific Blue. Titration curves are plotted as the mean fluorescence intensity (MFI) ± SD from three independent experiments ( n = 3). K D : equilibrium dissociation constant; n.a.: not applicable.

Article Snippet: To study the binding of FAPI TMs to hFAP, 96-well flat bottom plates were coated with 0.1 μg per well of recombinant hFAP protein (#3715-SE, R&D Systems).

Techniques: Binding Assay, Expressing, Flow Cytometry, In Vitro, Incubation, Titration, Fluorescence

Journal: Oncoimmunology

Article Title: Novel immunotheranostic FAP-inhibitor target modules for imaging and elimination of FAP-positive cells by UniCAR T-cells

doi: 10.1080/2162402X.2025.2598908

Figure Lengend Snippet: Evaluation of cytotoxicity and immunostimulatory cytokine production by UniCAR T-cells redirected by FAPI TMs. UniCAR T-cells were cultivated together with HT1080 hFAP or MRC-5 for 16 h in the absence (dark gray) or presence of NODA-GA-FAPI-PEG 12 -E5B9 (blue) or NODA-GA-FAPI-PEG 24 -E5B9 (green) TM. Target cell lysis was measured using luciferase-based assays. UniCAR T-cells were incubated with target cells at varying E:T ratios in the absence or presence of NODA-GA-FAPI-PEG 12 -E5B9 or NODA-GA-FAPI-PEG 24 -E5B9 TM (a, b). Titration curves were obtained for UniCAR T-cells co-cultured with target cells at an E:T ratio of 5:1. The dotted lines indicate the level of target cell lysis in the absence of FAPI TM (c, d). The statistical significance of the differences between the dose‒response curves was determined by nonlinear regression analysis using the extra sum-of-squares F-test in GraphPad Prism. Cytokines released by UniCAR T-cells cultivated with target cells and FAPI-TMs were quantified by ELISA (e, f). All results are plotted as the mean ± SD from three independent donors and experiments ( n = 3), each performed with three technical replicates. EC 50 : half-maximal effective concentration.

Article Snippet: To study the binding of FAPI TMs to hFAP, 96-well flat bottom plates were coated with 0.1 μg per well of recombinant hFAP protein (#3715-SE, R&D Systems).

Techniques: Lysis, Luciferase, Incubation, Titration, Cell Culture, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Oncoimmunology

Article Title: Novel immunotheranostic FAP-inhibitor target modules for imaging and elimination of FAP-positive cells by UniCAR T-cells

doi: 10.1080/2162402X.2025.2598908

Figure Lengend Snippet: Assessment of in vivo killing of UniCAR T-cells redirected by NODA-GA-FAPI-PEG 12/24 -E5B9 TMs. Female NXG mice were injected with either HT1080 hFAP cells (black), HT1080 hFAP and UniCAR T-cells (gray), or HT1080 hFAP and UniCAR T-cells at an E:T ratio of 1:1 in combination with 1 nmol of either NODA-GA-FAPI-PEG 12 -E5B9 (blue) or NODA-GA-FAPI-PEG 24 -E5B9 (green). The bioluminescence signal of HT1080 hFAP cells across all groups is shown for days 0 (1 h post-injection), 1, 2, 3 and 6 d post-injection (a). Quantitative analysis of the bioluminescence was conducted, and the results were plotted as mean ± SD from five mice per group ( n = 5) (b). Statistical significance was assessed using two-way ANOVA with Greenhouse–Geisser correction and with Dunett ´ s multiple comparison test with respect to the control group injected with only HT1080 hFAP cells. Stars indicate statistical significance; p -values close to the cutoff (0.05) are shown on the graph.

Article Snippet: To study the binding of FAPI TMs to hFAP, 96-well flat bottom plates were coated with 0.1 μg per well of recombinant hFAP protein (#3715-SE, R&D Systems).

Techniques: In Vivo, Injection, Comparison, Control

Journal: Oncoimmunology

Article Title: Novel immunotheranostic FAP-inhibitor target modules for imaging and elimination of FAP-positive cells by UniCAR T-cells

doi: 10.1080/2162402X.2025.2598908

Figure Lengend Snippet: Binding characteristics and pharmacokinetics of 64 Cu-radiolabeled FAPI TMs with different PEG spacer lengths. Maximum intensity projection (MIP) of NRMI nude mice bearing HT1080 (left flank, dashed white circles) and HT1080 hFAP tumor (right flank, white circles) after i.v. injection of 64 Cu-radiolabeled FAPI TMs ( a ). Time–activity curves (TAC, n = 2–3 mice per group) were analyzed for HT1080 hFAP tumor (b) and kidney (c) to determine tumor-to-kidney ratios over time (d). *: free urine.

Article Snippet: To study the binding of FAPI TMs to hFAP, 96-well flat bottom plates were coated with 0.1 μg per well of recombinant hFAP protein (#3715-SE, R&D Systems).

Techniques: Binding Assay, Drug discovery, Injection, Activity Assay

Journal: iScience

Article Title: The matricellular protein ADAMTS-like 2 regulates differentiation of skeletal muscle-resident fibro-adipogenic progenitor cells

doi: 10.1016/j.isci.2025.112712

Figure Lengend Snippet: FAP isolation and phenotypes of ADAMTSL2-deficient FAPs (A) Schematic of the magnetic activated cell sorting (MACS)-based FAP purification protocol. (B) PDGFRα immunostaining showing purity of FAP preparation from wild-type mice. Nuclei were counterstained with DAPI. (C) Adamtsl2 mRNA quantification in Ctrl and CKO-Prx FAPs after 4 days of fibrogenic differentiation. Adamtsl2 mRNA levels were normalized to Gapdh and Ctrl. (D) Western blot analysis of ADAMTSL2 protein in cell lysates from Ctrl and CKO-Prx FAPs. (E) Quantification of ADAMTSL2 band intensity in D normalized to GAPDH and Ctrl ( n = 3 independent FAP preparations). (F) Western blot analysis of ADAMTSL2 protein in cell lysates from primary Ctrl and CKO-Prx myoblasts isolated via pre-plating. (G) Ki67 immunostaining identifies proliferating FAPs isolated from Ctrl or CKO-Prx. Nuclei were counterstained with DAPI. (H) Quantification of percentage of proliferating Ki67+ cells from (G). n = 6 fields of view from n = 1 FAP isolate. (I) Schematic depicting the setup of the FAP/C2C12 myoblast co-culture system. (J) Visualization of multinucleated myotubes by immunostaining for myosin heavy chain (MyHC) and DAPI staining for nuclei. sh- Adamtsl2 -expressing C2C12 myoblasts were co-cultured with Ctrl or CKO-Prx FAPs. sh-Ctrl and sh- Adamtsl2 treated C2C12 myoblasts in single cultures served as controls for myotube formation and the effect of ADAMTSL2-depletion in myoblast differentiation as reported previously. (K) Quantification of fusion index, i.e., the percentage of nuclei in multinucleated myotubes, from (J). n = 3 co-cultures. Scale bars in (B), (G), and (J) represent 50 μm. Data are represented as mean ± SD. Statistical significance in (C), (E), and (H) was calculated with a two-sided Student’s t test and in (K) with a one-way ANOVA with posthoc-Tukey test. ∗ p < 0.05 and ∗∗∗ p < 0.001.

Article Snippet: Frozen primary human FAPs (hFAPs, FP-1111, F01743-63F.0.2.P2) were provided by Cook MyoSite. hFAPs were isolated from the vastus lateralis muscle using a proprietary process capitalizing on their surface expression of the PDGFRα antigen. hFAPs once grown were checked for purity utilizing anti-desmin to detect myogenic cell contaminants.

Techniques: Isolation, FACS, Purification, Immunostaining, Western Blot, Co-Culture Assay, Staining, Expressing, Cell Culture

Journal: iScience

Article Title: The matricellular protein ADAMTS-like 2 regulates differentiation of skeletal muscle-resident fibro-adipogenic progenitor cells

doi: 10.1016/j.isci.2025.112712

Figure Lengend Snippet: Adipogenic and fibrogenic differentiation of human FAPs (hFAPs) is regulated by recombinant ADAMTSL2 (A) Top: diagram of adipogenic hFAP differentiation. Bottom: brightfield images of hFAPs undergoing adipogenic differentiation. Inset shows an adipocyte with lipid droplets. (B) Relative quantification of ADAMTSL2 and perilipin ( PLIN1 ) mRNA isolated from hFAPs undergoing adipogenic differentiation. The fold-change after normalization to GAPDH expression is shown ( n = 3). (C) Perilipin immunostaining of hFAPs differentiated in the presence or absence of 30 μg (100 μg/mL) exogenous recombinant ADAMTSL2 (rL2) protein. Nuclei were counterstained with DAPI. (D) Quantification of perilipin MFI from (C). 2 fields of view from n = 4 wells per FAP isolate were analyzed. (E) Top: diagram of fibrogenic hFAP differentiation. Bottom: brightfield images of hFAPs undergoing fibrogenic differentiation. Inset shows elongated fibroblast-like cell morphology. (F) Relative quantification of ADAMTSL2 and ACTA2 (aSMA) mRNA isolated from hFAPs undergoing fibrogenic differentiation. The fold-change after normalization to GAPDH expression is shown ( n = 3). (G) aSMA immunostaining of hFAPs differentiated in the presence or absence of 30 μg (100 μg/mL) exogenous recombinant ADAMTSL2 (rL2) protein. Nuclei were counterstained with DAPI. (H) Quantification of αSMA MFI from (G). 2 fields of view from n = 4 wells per FAP isolate were analyzed. Scale bars in (A), (C), (E), and (G) represent 100 μm. Data are represented as mean ± SD. Statistical significance in (B) and (F) was calculated with a one-way ANOVA with posthoc-Tukey test and in (D) and (H) with a two-sided Student’s t test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: Frozen primary human FAPs (hFAPs, FP-1111, F01743-63F.0.2.P2) were provided by Cook MyoSite. hFAPs were isolated from the vastus lateralis muscle using a proprietary process capitalizing on their surface expression of the PDGFRα antigen. hFAPs once grown were checked for purity utilizing anti-desmin to detect myogenic cell contaminants.

Techniques: Recombinant, Quantitative Proteomics, Isolation, Expressing, Immunostaining

Journal: Oncology Reports

Article Title: Angiopoietin-like protein 2 induces androgen-independent and malignant behavior in human prostate cancer cells

doi: 10.3892/or.2014.3586

Figure Lengend Snippet: Expression of angiopoietin-like proteins (ANGPTLs) messenger RNA (mRNA) and protein. (A) Expression of ANGPTL families in LNCaP cells was analyzed using quantitative reverse-transcription polymerase chain reaction (RT-qPCR). The expression of ANGPTL2 mRNA was significantly increased ( * p<0.05 vs. ANGPTL6). (B) ANGPTL2 mRNA and protein expression was confirmed using RT-qPCR and western blot analysis in LNCaP, PC-3, DU 145 and 22Rv1 cells ( * p=0.34, # p=0.20 vs. LNCaP). (C) Immunohistochemical staining of LNCaP cells. β-tubulin served as the control.

Article Snippet: After blocking with Blocking One-P solution (Nacalai Tesque Inc., Kyoto, Japan), the membranes were incubated with mouse monoclonal antibodies against β-tubulin at a dilution of 1:5,000 (Sigma-Aldrich Corp.) or rabbit polyclonal antibodies against ANGPTL2 at a dilution of 1:1,000 (Proteintech Group, Chicago, IL, USA), Caspase-9, Caspase-3, poly adenosine diphosphate-ribose polymerase (PARP), Bcl-2 at a dilution of 1:1,000 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), Bcl-xL, Bad, E-Cadherin, N-Cadherin, vimentin, snail, slug at a dilution of 1:1,000 (Cell Signaling Technology, Inc., Danvers, MA, USA) and integrin α5β1 at a dilution of 1:1,000 (Biorbyt Ltd.) at 4°C overnight.

Techniques: Expressing, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Immunohistochemical staining, Staining, Control

Journal: Oncology Reports

Article Title: Angiopoietin-like protein 2 induces androgen-independent and malignant behavior in human prostate cancer cells

doi: 10.3892/or.2014.3586

Figure Lengend Snippet: Angiopoietin-like proteins (ANGPTL2) expression levels in LNCaP/AI cells. (A–D) LNCaP/AI cells showed significantly increased cell proliferation, migration and invasion compared with LNCaP cells (A, * p<0.05, ** p<0.03, # p=0.02, B, * p<0.05, ** p<0.01, # p<0.001, C, D, * p<0.05). (E and F) Quantitative reverse transcription polymerase chain reaction (RT-qPCR) and western blot analysis revealed that ANGPTL2 was significantly more expressed in LNCaP/AI cells than in LNCaP cells ( * p<0.05). β-tubulin.served as the control.

Article Snippet: After blocking with Blocking One-P solution (Nacalai Tesque Inc., Kyoto, Japan), the membranes were incubated with mouse monoclonal antibodies against β-tubulin at a dilution of 1:5,000 (Sigma-Aldrich Corp.) or rabbit polyclonal antibodies against ANGPTL2 at a dilution of 1:1,000 (Proteintech Group, Chicago, IL, USA), Caspase-9, Caspase-3, poly adenosine diphosphate-ribose polymerase (PARP), Bcl-2 at a dilution of 1:1,000 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), Bcl-xL, Bad, E-Cadherin, N-Cadherin, vimentin, snail, slug at a dilution of 1:1,000 (Cell Signaling Technology, Inc., Danvers, MA, USA) and integrin α5β1 at a dilution of 1:1,000 (Biorbyt Ltd.) at 4°C overnight.

Techniques: Expressing, Migration, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Control

Journal: Oncology Reports

Article Title: Angiopoietin-like protein 2 induces androgen-independent and malignant behavior in human prostate cancer cells

doi: 10.3892/or.2014.3586

Figure Lengend Snippet: Effects of angiopoietin-like proteins (ANGPTL2) small-interfering RNA (siRNA) on cell proliferation, migration and invasion in LNCaP cells. (A) ANGPTL2 siRNA downregulated ANGPTL2 expression for 72 h after transfection into LNCaP cells. (B) LNCaP/si-ANGPTL2 cell growth was more restricted than in LNCaP/wild-type (WT) or LNCaP/si-Scr ( * p<0.05). (C) LNCaP/si-ANGPTL2 reduced cell migration and invasion significantly more than in LNCaP/WT or LNCaP/si-Scr ( * p<0.05).

Article Snippet: After blocking with Blocking One-P solution (Nacalai Tesque Inc., Kyoto, Japan), the membranes were incubated with mouse monoclonal antibodies against β-tubulin at a dilution of 1:5,000 (Sigma-Aldrich Corp.) or rabbit polyclonal antibodies against ANGPTL2 at a dilution of 1:1,000 (Proteintech Group, Chicago, IL, USA), Caspase-9, Caspase-3, poly adenosine diphosphate-ribose polymerase (PARP), Bcl-2 at a dilution of 1:1,000 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), Bcl-xL, Bad, E-Cadherin, N-Cadherin, vimentin, snail, slug at a dilution of 1:1,000 (Cell Signaling Technology, Inc., Danvers, MA, USA) and integrin α5β1 at a dilution of 1:1,000 (Biorbyt Ltd.) at 4°C overnight.

Techniques: Small Interfering RNA, Migration, Expressing, Transfection

Journal: Oncology Reports

Article Title: Angiopoietin-like protein 2 induces androgen-independent and malignant behavior in human prostate cancer cells

doi: 10.3892/or.2014.3586

Figure Lengend Snippet: Effects of angiopoietin-like proteins (ANGPTL2) small interfering RNA (siRNA) on cell proliferation, migration and invasion in LNCaP/AI cells. (A) ANGPTL2 siRNA downregulated ANGPTL2 expression after transfection into LNCaP/AI cells. (B) LNCaP/AI/si-ANGPTL2 cell growth was significantly more restricted than that in LNCaP/AI/WT or LNCaP/AI/si-Scr ( * p<0.05, # p<0.02). (C) LNCaP/AI/si-ANGPTL2 more significantly reduced cell migration and invasion than that in LNCaP/AI/WT or LNCaP/AI/si-Scr ( * p<0.05). (D) Activation of caspase-9 and caspase-3 and poly adenosine diphosphate-ribose (PARP) cleavage after transfection with ANGPTL2 siRNA or si-Scr were determined using western blot analysis. Treatment with ANGPTL2 siRNA induced caspase-9 and caspase-3 activation and PARP cleavage in LNCaP/AI cells.

Article Snippet: After blocking with Blocking One-P solution (Nacalai Tesque Inc., Kyoto, Japan), the membranes were incubated with mouse monoclonal antibodies against β-tubulin at a dilution of 1:5,000 (Sigma-Aldrich Corp.) or rabbit polyclonal antibodies against ANGPTL2 at a dilution of 1:1,000 (Proteintech Group, Chicago, IL, USA), Caspase-9, Caspase-3, poly adenosine diphosphate-ribose polymerase (PARP), Bcl-2 at a dilution of 1:1,000 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), Bcl-xL, Bad, E-Cadherin, N-Cadherin, vimentin, snail, slug at a dilution of 1:1,000 (Cell Signaling Technology, Inc., Danvers, MA, USA) and integrin α5β1 at a dilution of 1:1,000 (Biorbyt Ltd.) at 4°C overnight.

Techniques: Small Interfering RNA, Migration, Expressing, Transfection, Activation Assay, Western Blot

Journal: Oncology Reports

Article Title: Angiopoietin-like protein 2 induces androgen-independent and malignant behavior in human prostate cancer cells

doi: 10.3892/or.2014.3586

Figure Lengend Snippet: Effects of angiopoietin-like proteins (ANGPTL2) overexpression on cell proliferation, migration and invasion in LNCaP cells. (A) Human ANGPTL2 cDNA expression vector upregulated ANGPTL2 expression following transfection into LNCaP cells. (B), (C) ANGPTL2-overexpressing transfectant growth rates were significantly higher than those of LNCaP/WT or LNCaP/Neo in the general and androgen-deprived media (B, * p<0.02, ** p<0.02, # p<0.01, C, * p<0.01, ** p<0.01, # p<1.0E-07). (D) LNCaP/ANGPTL2 has upregulated cell growth for 10 and 20 days in androgen-deprived medium.

Article Snippet: After blocking with Blocking One-P solution (Nacalai Tesque Inc., Kyoto, Japan), the membranes were incubated with mouse monoclonal antibodies against β-tubulin at a dilution of 1:5,000 (Sigma-Aldrich Corp.) or rabbit polyclonal antibodies against ANGPTL2 at a dilution of 1:1,000 (Proteintech Group, Chicago, IL, USA), Caspase-9, Caspase-3, poly adenosine diphosphate-ribose polymerase (PARP), Bcl-2 at a dilution of 1:1,000 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), Bcl-xL, Bad, E-Cadherin, N-Cadherin, vimentin, snail, slug at a dilution of 1:1,000 (Cell Signaling Technology, Inc., Danvers, MA, USA) and integrin α5β1 at a dilution of 1:1,000 (Biorbyt Ltd.) at 4°C overnight.

Techniques: Over Expression, Migration, Expressing, Plasmid Preparation, Transfection

Journal: Oncology Reports

Article Title: Angiopoietin-like protein 2 induces androgen-independent and malignant behavior in human prostate cancer cells

doi: 10.3892/or.2014.3586

Figure Lengend Snippet: Cell migration and invasion of the angiopoietin-like proteins (ANGPTL2)-overexpressing transfectants and expressions of apoptosis and epithelial-to-mesenchymal transition (EMT)-associated proteins. (A) LNCaP/ANGPTL2 showed significantly increased cell migration and invasion than that in LNCaP/WT or LNCaP/Neo ( * p<0.01). (B) LNCaP/ANGPTL2 increased the expression levels of Bcl-2 and Bcl-xL proteins and decreased the expression level of Bad protein. (C) LNCaP/ANGPTL2 increased the expression levels of snail, slug, N-Cadherin and vimentin proteins and decreased the expression level of E-Cadherin protein.

Article Snippet: After blocking with Blocking One-P solution (Nacalai Tesque Inc., Kyoto, Japan), the membranes were incubated with mouse monoclonal antibodies against β-tubulin at a dilution of 1:5,000 (Sigma-Aldrich Corp.) or rabbit polyclonal antibodies against ANGPTL2 at a dilution of 1:1,000 (Proteintech Group, Chicago, IL, USA), Caspase-9, Caspase-3, poly adenosine diphosphate-ribose polymerase (PARP), Bcl-2 at a dilution of 1:1,000 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), Bcl-xL, Bad, E-Cadherin, N-Cadherin, vimentin, snail, slug at a dilution of 1:1,000 (Cell Signaling Technology, Inc., Danvers, MA, USA) and integrin α5β1 at a dilution of 1:1,000 (Biorbyt Ltd.) at 4°C overnight.

Techniques: Migration, Expressing

Journal: Oncology Reports

Article Title: Angiopoietin-like protein 2 induces androgen-independent and malignant behavior in human prostate cancer cells

doi: 10.3892/or.2014.3586

Figure Lengend Snippet: Integrin α5β1 expression levels in LNCaP and LNCaP/AI cells. Effects of integrin α5β1 immunoneutralization and angiopoietin-like (ANGPTL2) recombinant protein on cell migration in LNCaP cells. (A and B) Expression of integrin α5β1 protein in LNCaP and LNCaP/AI cells was confirmed using western blot analysis and immunohistochemical staining. (C) Cell migration with integrin α5β1 immunoneutralization was significantly more restricted than that in LNCaP/WT or LNCaP/control mouse IgG. Migration rates were increased significantly depending on the concentration of ANGPTL2 recombinant protein ( * p<0.05 vs. anti-integrin α5β1(−). ** p<0.05 vs. ANGPTL2 (5 μg/ml), anti-integrin α5β1(−). # p<0.05 vs. ANGPTL2 (0 μg/ml). ## p<0.01 vs. ANGPTL2 (0 μg/ml).

Article Snippet: After blocking with Blocking One-P solution (Nacalai Tesque Inc., Kyoto, Japan), the membranes were incubated with mouse monoclonal antibodies against β-tubulin at a dilution of 1:5,000 (Sigma-Aldrich Corp.) or rabbit polyclonal antibodies against ANGPTL2 at a dilution of 1:1,000 (Proteintech Group, Chicago, IL, USA), Caspase-9, Caspase-3, poly adenosine diphosphate-ribose polymerase (PARP), Bcl-2 at a dilution of 1:1,000 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), Bcl-xL, Bad, E-Cadherin, N-Cadherin, vimentin, snail, slug at a dilution of 1:1,000 (Cell Signaling Technology, Inc., Danvers, MA, USA) and integrin α5β1 at a dilution of 1:1,000 (Biorbyt Ltd.) at 4°C overnight.

Techniques: Expressing, Recombinant, Migration, Western Blot, Immunohistochemical staining, Staining, Control, Concentration Assay

Journal: Oncology Reports

Article Title: Angiopoietin-like protein 2 induces androgen-independent and malignant behavior in human prostate cancer cells

doi: 10.3892/or.2014.3586

Figure Lengend Snippet: Angiopoietin-like proteins (ANGPTL2) expression levels in human prostate cancer tissues. (A) Prostate cancer tissue without androgen ablation therapy (Gleason score 3+3=6, well differentiated). (B) Prostate cancer tissue after androgen ablation therapy (Gleason score 3+4=7, well differentiated). Weakly positive staining of ANGPTL2 was observed in specimens obtained from patients without neoadjuvant hormonal therapy. However surviving prostate cancer cells were strongly positive-staining after hormonal therapy.

Article Snippet: After blocking with Blocking One-P solution (Nacalai Tesque Inc., Kyoto, Japan), the membranes were incubated with mouse monoclonal antibodies against β-tubulin at a dilution of 1:5,000 (Sigma-Aldrich Corp.) or rabbit polyclonal antibodies against ANGPTL2 at a dilution of 1:1,000 (Proteintech Group, Chicago, IL, USA), Caspase-9, Caspase-3, poly adenosine diphosphate-ribose polymerase (PARP), Bcl-2 at a dilution of 1:1,000 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), Bcl-xL, Bad, E-Cadherin, N-Cadherin, vimentin, snail, slug at a dilution of 1:1,000 (Cell Signaling Technology, Inc., Danvers, MA, USA) and integrin α5β1 at a dilution of 1:1,000 (Biorbyt Ltd.) at 4°C overnight.

Techniques: Expressing, Staining