Journal: ACS Chemical Neuroscience

Article Title: Ester Prodrug NLRP3 Inflammasome Inhibitor NT-0796 is Brain Active due to Activation by Local Expression of Carboxylesterase‑1

doi: 10.1021/acschemneuro.5c00843

Figure Lengend Snippet: Evidence of NT-0796 hydrolysis in NHP brain homogenate. (A) NT-0796 (3 μM) was incubated with brain supernatant fraction (#1), CES1 supersomes, or buffer alone. At the indicated times, aliquots of the reaction mixtures were quenched, and concentrations of NDT-19795 were determined by LC-MS/MS analysis and indicated as a function of treatment and time at 37 °C. (B) NHP brain homogenate supernatant fractions #1 and #2, along with CES1 supersomes, were treated with FP-biotin, after which tagged proteins were subsequently captured with streptavidin agarose. After dissociation, the isolates were separated by SDS gel electrophoresis and transferred to nitrocellulose. The blot was stained sequentially with Alexafluor 680-conjugated streptavidin (red) and rabbit anti-CES1, followed by Alexafluor 800-conjugated goat-antirabbit secondary antibody (green). Lanes of the blot correspond to (1) FP-biotin-treated NHP brain supernatant fraction #1 (200 μg), (2) FP-biotin treated NHP brain supernatant fraction #2 (9 μg), (3) FP-biotin-treated CES1 supersomes (3 μg), (4) NHP brain supernatant fraction #1 (200 μg), (5) NHP brain supernatant fraction #2 (9 μg), (6) CES1 supersomes (3 μg), (7) blank, and (8) molecular weight markers (size indicated on the right). The blot was simultaneously imaged with both 700 and 800 nm lasers, allowing simultaneous visualization of both fluorescent tags.

Article Snippet: Second, the membranes were probed with streptavidin conjugated to Alexafluor 680 (Thermo Fisher, S21378; diluted 1:4000 in blocking buffer containing 1% Tween 20) for 1 h at room temperature, followed by multiple washes with PBS, 0.05% Tween 20.

Techniques: Incubation, Liquid Chromatography with Mass Spectroscopy, SDS-Gel, Electrophoresis, Staining, Molecular Weight

Journal: Life

Article Title: Bioactivity Potential of Bioceramic-Based Root Canal Sealers: A Scoping Review

doi: 10.3390/life12111853

Figure Lengend Snippet: Studies evaluating the potential for cellular bioactivity in vitro.

Article Snippet: [ ] Tanomaru-Filho et al., 2017 , International Endodontic Journal , Saos-2 human osteoblast-like cells (ATCC HTB-85) , experimental TSC/Ta2O5, Neo MTA Plus (Avalon Biomed Inc.), MTA (Angelus) , 1: 8 dilution , Assess biocompatibility and formation of mineralized nodules , ALP a (kit comercial) , (18 per group) , 1, 3, and 7 days , All materials induced the production of mineralized nodules, being higher with MTA Plus..

Techniques: Activity Assay, Staining, Enzyme-linked Immunosorbent Assay, Enzymatic Assay, Microscopy, Ex Vivo, Control, Immunofluorescence, Expressing, Sequencing, Epifluorescence Microscopy, Immunocytochemistry, Fluorescence, Gene Expression, Cell Culture, In Vitro, Tube Formation Assay, Extraction, Real-time Polymerase Chain Reaction, Marker, Spectrophotometry, Migration, Cell Attachment Assay, Positive Control, Produced, Spectroscopy, Negative Control, Quantitative RT-PCR, Western Blot, RNA Expression

Journal: Cancer Discovery

Article Title: PTEN Loss Promotes PI3Kβ Phosphorylation and EPHA2/SRC/p-PI3Kβ Y962 Complex Assembly to Drive Tumorigenesis

doi: 10.1158/2159-8290.CD-25-1126

Figure Lengend Snippet: BioID protein interaction profiling reveals a PI3Kβ–EPHA2 interaction induced by PTEN loss in cancer cells. A, Workflow depicting the BioID experiment. PTEN-null cancer cells were overexpressed with vector, BioID- PIK3CA , or BioID- PIK3CB , and biotin was added to the culture medium, followed by immunoprecipitating with SBP beads. Biotinylation and identification of bait-interacting protein was detected by MS. B, Heatmap showing the MS intensities of all proteins pulled down by PIK3CA -BirA* or PIK3CB -BirA* in PTEN-null cell lines (MDA-MB-468 and BT549). Clustering was assessed with the Euclidean distance measurement in column and the Z-normalization in row. Proteins of interest were colored. C, Plot of sum intensity for protein interactors of PI3Kα and PI3Kβ identified by MS analysis from cells expressing PIK3CA-BirA* or PIK3CB-BirA* in PTEN-null cell lines (MDA-MB-468 and BT549). D, EPHA2 colocalized with PI3Kβ on the cell membrane. IF staining with anti-PI3Kβ (green) and anti-EPHA2 (red) in PTEN-null BT549 cells. DAPI was used for nuclear staining. Scale bars, 50 μm. E, Endogenous EPHA2 IP from PTEN-null BT549 and HCC70 cells analyzed by Western blot with the indicated antibodies. F, PTEN loss enhanced PI3Kβ–EPHA2 interaction. PTEN-WT and PTEN-KO HEK293T cells were transfected with Flag-tagged PI3Kα, PI3Kβ, and EPHA2-HA, and cell lysate was immunoprecipitated with anti-Flag antibody. G, PTEN restoration blunted the dominant interaction between EPHA2 and PI3Kβ. Co-IP assay of exogenous PI3Kα and PI3Kβ with exogenous EPHA2 in vector and PTEN stably overexpressed BT549 cells. Flag-tagged PI3Kα and PI3Kβ were immunoprecipitated with anti-Flag antibody and then analyzed by Western blot with the indicated antibodies. H, PTEN restoration blunted the dominant interaction between endogenous EPHA2 and PI3Kβ in BT549 cells.

Article Snippet: EPHA2 inhibitors NVP-BHG712 (MCE, cat. #HY-13258A, CAS No:940310-85-0)/ALW-II-41-27 (MCE, cat. #HY-18007, CAS No: 1186206-79-0) and ERK inhibitor GSK2656157 (MCE, cat. #HY-13820, CAS No: 1337532-29-2) were introduced into the culture medium 48 hours before cell harvest.

Techniques: Plasmid Preparation, Expressing, Membrane, Staining, Western Blot, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Stable Transfection

Journal: Cancer Discovery

Article Title: PTEN Loss Promotes PI3Kβ Phosphorylation and EPHA2/SRC/p-PI3Kβ Y962 Complex Assembly to Drive Tumorigenesis

doi: 10.1158/2159-8290.CD-25-1126

Figure Lengend Snippet: PTEN acts as a protein tyrosine phosphatase regulating PI3Kβ–EPHA2 interaction through dephosphorylation of tyrosine-962 (Y962) on PI3Kβ to impact tumorigenesis in vivo . A, Pan-phosphotyrosine of PI3Kβ was upregulated in PTEN-KO HEK293T cells. PTEN-WT and PTEN-KO HEK293T cells were immunoprecipitated with anti-PI3Kα and anti-PI3Kβ antibodies, respectively, followed by immunoblotting analysis with anti–pan-phosphotyrosine antibody. B and C, Phosphotase activity of PTEN dephosphorylated the tyrosine residues of PI3Kβ. BT549 cells and HCC70 cells stably expressing empty vector, PTEN-WT, PTEN G129E, Y138L, and C124S mutants were immunoprecipitated with anti-PI3Kβ antibody and then analyzed by Western blot with the indicated antibodies. D, PTEN reduced the PI3Kβ–EPHA2 interaction dependent on its protein phosphotase activity. PTEN-null BT549 cells stably expressing vector, PTEN-WT, PTEN G129E, Y138L, or C124S mutants were overexpressed with Flag-tagged PI3Kβ and EPHA2 and then immunoprecipitated with anti-Flag antibody and analyzed by Western blot with the indicated antibodies. E and F, Schematic of the phosphorylation MS ( E ). PTEN KO HEK293T cells were transfected with Flag-tagged PIK3CB . After harvesting, the cell lysate was immunoprecipitated with anti-Flag magnetic beads to purify Flag-tagged PI3Kβ. The MS/MS spectra that identified Y962 and T930 were phosphorylated on PI3Kβ in PTEN-KO HEK293T cells ( F ). G, Protein sequence alignments of PI3Kβ from indicated species based on amino acids surrounding the Y962 site of PI3Kβ protein. H, PTEN removed p-PI3Kβ Y962 peptide but not p-PI3KβT930 peptide in vitro . PTEN-WT and G129E, Y138L, and C124S mutants immunoprecipitated from HEK293T cells were incubated with synthetic phosphorylated PI3Kβ-Y962 or PI3Kβ-T930 peptides in dephosphorylation buffer, and the resulting peptides were analyzed by MS. I, Mimic nonphosphorylatable mutant at PI3Kβ-Y962 blunted its interaction with EPHA2 in PTEN-null cancer cells. BT549 cells were transfected with Flag-tagged human WT PIK3CB , T930A, Y962F, and T930A/Y962F mutants and HA-tagged EPHA2, and cell lysates were immunoprecipitated with anti-Flag antibody and analyzed by Western blot with the indicated antibodies. J, Mimic phosphorylation mutant at PI3Kβ-Y962 promoted its interaction with EPHA2 in PTEN-WT MD-MBA-231 cells. MD-MBA-231 cells were transfected with Flag-tagged human PI3Kβ-WT, PI3Kβ-T930E, -Y962E, and -T930E/Y962E mutants and EPHA2, and cell lysates were immunoprecipitated with anti-Flag antibody. K–N, Dephosphorylation mutant at mouse PI3Kβ-Y956 inhibited tumor growth of PPB xenografts, as measured by significantly reduced tumor growth curve, tumor volume, tumor weight, and Ki67 expression levels. ( K and M , n = 10 per group combining 2 mouse cohorts; L and N , n = 6 per group). O–R, Dephosphorylation mutant at human PI3Kβ-Y962 inhibited growth of PTEN-null PC3 tumors, as measured by significantly reduced tumor growth curve, tumor volume, tumor weight, and Ki67 expression levels ( n = 10 per group). Values were presented as the mean ± SEM. P values were determined by two-way ANOVA (K and O) and one-way ANOVA with Dunnett correction multiple-comparison test (M, N, Q, R). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: EPHA2 inhibitors NVP-BHG712 (MCE, cat. #HY-13258A, CAS No:940310-85-0)/ALW-II-41-27 (MCE, cat. #HY-18007, CAS No: 1186206-79-0) and ERK inhibitor GSK2656157 (MCE, cat. #HY-13820, CAS No: 1337532-29-2) were introduced into the culture medium 48 hours before cell harvest.

Techniques: De-Phosphorylation Assay, In Vivo, Immunoprecipitation, Western Blot, Activity Assay, Stable Transfection, Expressing, Plasmid Preparation, Phospho-proteomics, Transfection, Magnetic Beads, Tandem Mass Spectroscopy, Sequencing, In Vitro, Incubation, Mutagenesis, Comparison

Journal: Cancer Discovery

Article Title: PTEN Loss Promotes PI3Kβ Phosphorylation and EPHA2/SRC/p-PI3Kβ Y962 Complex Assembly to Drive Tumorigenesis

doi: 10.1158/2159-8290.CD-25-1126

Figure Lengend Snippet: Phosphorylation of PI3Kβ is critical for PTEN-null tumor formation and growth in vivo . A, Protein levels of PI3Kα, PI3Kβ, p-ERK, ERK, c-Myc, pAKT, AKT in WT, PIK3CA -KO, and PIK3CB -KO BT549 cells. B, Protein phosphatase activity of PTEN downregulated c-Myc. PTEN-null BT549 cells stably expressed vector, PTEN-WT, PTEN-G129E, -Y138L, and -C124S mutants, and whole-cell lysates were analyzed by Western blot with the indicated antibodies. C, PI3Kβ-Y962F or PI3Kβ-T930A/Y962F lost the ability of PI3Kβ to promote p-ERK and c-Myc together with decreased pAKT, whereas PI3Kβ-K805R lost the ability of PI3Kβ to promote pAKT in PTEN-null cancer cells. The indicated mutants of PI3Kβ were transfected to endogenous PI3Kβ-depleted BT549 cells, and protein levels were analyzed by Western blot with the indicated antibodies. D, EPHA2 knockdown decreased p-ERK and c-Myc in BT549 cells. E, PI3Kβ enhanced EPHA2 enzyme activity, whereas dephosphorylation of PI3Kβ-Y962 or PI3Kβ-T930/Y962 impairs the ability of PI3Kβ in activating EPHA2. The indicated mutants of PI3Kβ were transfected to endogenous PI3Kβ knockdown BT549 cells. Endogenous EPHA2 was immunoprecipitated, followed by EPHA2 kinase activity assay ( n = 9 per group). F and G, Either ERK knockdown or treatment with an ERK inhibitor blunted the enhanced c-Myc protein levels by addback of PI3Kβ in sh PIK3CB BT549 cells. Western blot analysis revealed reduced phosphorylation of c-Myc at S62, and c-Myc protein levels in cells treated with ERK knockdown or an ERK inhibitor after PI3Kβ reconstitution. H and I, Either ERK knockdown or c-MYC knockdown blunted the increased cell proliferation elicited by addback of PI3Kβ in sh PIK3CB BT549 cells. J, Phosphorylation status of PI3Kβ presented no impact on its classic lipid activity. Flag-tagged PI3Kβ-WT, -T930A, -Y962F, -T930A/Y962F, and -K805R mutants were immunoprecipitated from HEK293T cells, followed by lipid kinase activity assay ( n = 4 per group). K, Kinase activity of PI3Kβ presented no impact on PI3Kβ–EPHA2 interaction. PTEN-null BT549 cells and PTEN-KO HEK293T cells were transfected with Flag-tagged PI3Kβ-WT or Flag-tagged PI3Kβ-K805R (kinase-dead mutant) together with HA-tagged EPHA2. Cell lysate was immunoprecipitated with anti-Flag antibody. L, A model showing PTEN loss upregulates PI3Kβ-Y962 phosphorylation and strengthens the EPHA2–PI3Kβ interaction, triggering EPHA2 activation, ERK phosphorylation, and ultimately c-Myc stabilization. Moreover, although PI3Kβ phosphorylation showed no significant impact on its kinase activity, EPHA2–pPI3Kβ interaction might increase accessibility of PI3Kβ to phosphorylate PIP2 on the cell membrane to affect pAKT in PTEN-loss cancers. Values were presented as the mean ± SEM. P values were determined by two-way ANOVA (H and I) and one-way ANOVA with Dunnett correction multiple-comparison test (E and J). *, P < 0.05; **, P < 0.001; ****, P < 0.0001.

Article Snippet: EPHA2 inhibitors NVP-BHG712 (MCE, cat. #HY-13258A, CAS No:940310-85-0)/ALW-II-41-27 (MCE, cat. #HY-18007, CAS No: 1186206-79-0) and ERK inhibitor GSK2656157 (MCE, cat. #HY-13820, CAS No: 1337532-29-2) were introduced into the culture medium 48 hours before cell harvest.

Techniques: Phospho-proteomics, In Vivo, Activity Assay, Stable Transfection, Plasmid Preparation, Western Blot, Transfection, Knockdown, De-Phosphorylation Assay, Immunoprecipitation, Kinase Assay, Mutagenesis, Activation Assay, Membrane, Comparison

Journal: Cancer Discovery

Article Title: PTEN Loss Promotes PI3Kβ Phosphorylation and EPHA2/SRC/p-PI3Kβ Y962 Complex Assembly to Drive Tumorigenesis

doi: 10.1158/2159-8290.CD-25-1126

Figure Lengend Snippet: A SRC–EPHA2–PI3Kβ tripartite complex drives oncogenic signaling in PTEN-null tumors. A, A schematic of the experiment for the targeted compound library screening to identify PI3Kβ phosphorylation inhibitors. Endogenous PI3Kβ-depleted BT549 or PC3 cells replaced with PI3Kβ-WT or PI3Kβ-Y962F mutants were treated with compounds from the library at 0.1 μmol/L. Cell viability was assessed by CCK-8 assays at 48 hours. B and C, Results of the kinase inhibitor screening in BT549 ( B ) and PC3 ( C ) cells. SRC inhibitors dasatinib and KX2-391 were among the top three significant inhibitors in both cells. D and E, Dasatinib effectively inhibited p-SRC, p-PI3Kβ Y962 , p-ERK/c-Myc, and pAKT in PTEN-null BT549 and PC3 cells. F, KX2-391 effectively inhibited p-SRC, p-PI3Kβ Y962 , p-ERK/c-Myc, and pAKT in PTEN-null BT549 cells. G, Dasatinib and KX2-391 dramatically reduced the growth of PPB cells with addback of PI3Kβ-WT but was not as effective on PPB cells with addback of PI3Kβ-Y956F. H, Knockdown of SRC or EPHA2 decreased phosphorylation of PI3Kβ-Y962 and c-MYC in BT549 cells. I, SRC coordinated with EPHA2 to phosphorylate PI3Kβ. SRC-Flag and EPHA2 plasmids were overexpressed in PTEN-WT and PTEN-KO HEK293T cells, and Flag was immunoprecipitated, followed by Western blot analysis. J, SRC and EPHA2 phosphorylated PI3Kβ-Y962 peptides in vitro . GST-SRC or GST-EPHA2 was incubated with synthetic Y962 containing PI3Kβ peptides in phosphorylation buffer, and the resulting peptides were analyzed by MS spectrum. K, In vitro phosphorylation assays showed that SRC and EPHA2 directly phosphorylated PI3Kβ-Y962, and the phosphorylation activity was blunted on PI3Kβ mutant at Y962. Particularly, SRC could strongly phosphorylate PI3Kβ-Y962. An in vitro kinase assay was performed by mixing GST-SRC or GST-EPHA2 with Flag-PI3Kβ-WT or Flag-PI3Kβ-Y962F proteins in the presence of ATP. Anti–phospho-PI3Kβ-Y962 antibody was used to detect the phosphorylated PI3Kβ-Y962. L, Knockdown of SRC (shSRC) dramatically decreased signaling of the p-PI3Kβ Y962 –pERK/c-Myc axis in BT549 cells; this effect can be slightly rescued by exogenous expression of EPHA2. M, Knockdown of EPHA2 (shEPHA2) decreased signaling of the p-PI3Kβ Y962 –pERK/c-Myc axis in BT549 cells; exogenous expression of SRC was unable to rescue the effect by sh EPHA2 in BT549 cells. N, SRC expression elevated p-ERK and c-Myc levels in BT549 cells, which were abolished by EPHA2 KO. O, A mechanism model to show that SRC collaborates with EPHA2 to phosphorylate PI3Kβ-Y962, whereas PTEN loss abolished PI3Kβ dephosphorylation, leading to PI3Kβ hyperphosphorylation and subsequent SRC–EPHA2–p-PI3Kβ Y962 complex formation to upregulate p-ERK/c-Myc signaling, accompanying with enhanced accessibility of PI3Kβ to phosphorylate PIP2 on the cell membrane to upregulate pAKT. Values were presented as the mean ± SEM. P values were determined by unpaired two-tailed t test (B, C). **** P < 0.0001.

Article Snippet: EPHA2 inhibitors NVP-BHG712 (MCE, cat. #HY-13258A, CAS No:940310-85-0)/ALW-II-41-27 (MCE, cat. #HY-18007, CAS No: 1186206-79-0) and ERK inhibitor GSK2656157 (MCE, cat. #HY-13820, CAS No: 1337532-29-2) were introduced into the culture medium 48 hours before cell harvest.

Techniques: Drug discovery, Phospho-proteomics, CCK-8 Assay, Knockdown, Immunoprecipitation, Western Blot, In Vitro, Incubation, Activity Assay, Mutagenesis, Kinase Assay, Expressing, De-Phosphorylation Assay, Membrane, Two Tailed Test

Journal: Cancer Discovery

Article Title: PTEN Loss Promotes PI3Kβ Phosphorylation and EPHA2/SRC/p-PI3Kβ Y962 Complex Assembly to Drive Tumorigenesis

doi: 10.1158/2159-8290.CD-25-1126

Figure Lengend Snippet: Pharmacologically blocking p-PI3Kβ Y962 using dasatinib exhibits potent antitumor efficacy against multiple PTEN-deficient tumors in vivo . A–C, Dasatinib treatment significantly inhibited PP tumor growth in vivo , decreasing tumor growth and tumor weight. ( n = 10 per group). D, Dasatinib treatment reduced PI3Kβ–EPHA2 interaction in PP tumors. Endogenous EPHA2 was pulled down and co-immunoprecipitated with PI3Kβ in PP tumors with vehicle or dasatinib treatment. E, Dasatinib treatment reduced p-PI3Kβ Y962 and c-Myc in PP tumors, as measured by Western blots of the tumors under vehicle or dasatinib with p-PI3Kβ Y962 and c-Myc antibody. F–I, Dasatinib treatment reduced p-PI3Kβ Y962 , c-Myc, and Ki67 in PP tumors, as measured by IHC staining. Representative IHC images of p-PI3Kβ Y962 , c-Myc, and ki67 staining were shown ( I ). Scale bar, 50 μm. J, Dasatinib treatment significantly inhibited growth of PTEN-null human HCC70 xenografts. Tumor weight and c-MYC levels of HCC70 tumors at the end of treatment with vehicle or dasatinib were shown. ( n = 6 or 10 per group). K, Dasatinib treatment showed no significant effect on the growth of PTEN-WT human HCC1954 xenografts. Tumor weight and c-MYC levels of HCC1954 tumors at the end of treatment with vehicle or dasatinib were shown. ( n = 4 or 6 per group). L–N, Dasatinib treatment inhibited tumor growth in human TNBC PDX models carrying PTEN deletion. Tumor growth curve, tumor weight, and tumor volumes of DFBC-1613 PDX upon treatment with vehicle or dasatinib were shown. ( n = 8 per group). O and P, Representative IHC images and IHC score analysis of c-Myc staining in DFCI-1613 PDX tumors. Scale bar, 50 μm. Q, A phosphorylation-based mechanism of PI3Kβ activation that plays a critical role in PTEN-null tumorigenesis. PTEN loss leads to PI3Kβ-Y962 hyperphosphorylation and formation of an EPHA2–SRC–PI3Kβ complex, upregulating p-ERK/c-Myc and pAKT signaling to strongly drive the development and progression of PTEN-deficient tumors. Importantly, phosphorylation of PI3Kβ Y962 is a functional biomarker and druggable therapeutic target for PTEN-deficient tumors. The violin plots showed all points, and P values were determined by nonparametric Mann–Whitney test. The values of tumor growth curves were presented as the mean ± SEM, using two-way ANOVA test for statistical analysis. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: EPHA2 inhibitors NVP-BHG712 (MCE, cat. #HY-13258A, CAS No:940310-85-0)/ALW-II-41-27 (MCE, cat. #HY-18007, CAS No: 1186206-79-0) and ERK inhibitor GSK2656157 (MCE, cat. #HY-13820, CAS No: 1337532-29-2) were introduced into the culture medium 48 hours before cell harvest.

Techniques: Blocking Assay, In Vivo, Immunoprecipitation, Western Blot, Immunohistochemistry, Staining, Phospho-proteomics, Activation Assay, Functional Assay, Biomarker Discovery, MANN-WHITNEY

Journal: Communications Biology

Article Title: Single-stranded DNA in the bone microenvironment promotes prostate cancer bone metastasis via the ITGA6-FAK pathway

doi: 10.1038/s42003-026-09929-9

Figure Lengend Snippet: A Representative BLI images from mice in the Vehicle and Defactinib groups. B Kaplan–Meier survival curve of mice in Vehicle, Defactinib, Vehicle + EHBP1-ssDNA, and Defactinib + EHBP1-ssDNA groups ( n = 8). C Representative BLI images from mice in Vehicle + EHBP1-ssDNA and Defactinib + EHBP1-ssDNA groups. D Quantification of the BLI signals of bone metastases in each group ( n = 8). E Representative micro-CT 3D reconstructed images from mice in Vehicle and Defactinib groups. F Quantitative micro-CT analysis of the trabecular bone microarchitecture of mice femurs from Vehicle and Defactinib groups ( n = 8). G Representative micro-CT 3D reconstructed images from mice in Vehicle + EHBP1-ssDNA and Defactinib + EHBP1-ssDNA groups. H Quantitative micro-CT analysis of the trabecular bone microarchitecture of mice femurs from Vehicle + EHBP1-ssDNA and Defactiib + EHBP1-ssDNA groups ( n = 8). I Representative H&E images and quantification of relative tumor area from mice in Vehicle and Defactinib groups ( n = 8). Scale bars represent 100 μm. J Representative H&E images and quantification of relative tumor area from mice in Vehicle + EHBP1-ssDNA and Defactinib + EHBP1-ssDNA groups ( n = 8). Scale bars represent 100 μm. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Student’s t -test was used for ( D , F , H , I , J ). A two-sided log-rank test was used for the survival difference in ( B ). The error bar represent standard error of the mean. BV/TV trabecular bone volume per tissue volume, Tb.Th trabecular thickness.

Article Snippet: Defactinib (MedChemExpress, 1073154-85-4) at 25 mg/kg was administered via oral gavage once a day for 4 weeks.

Techniques: Micro-CT

Journal: Communications Biology

Article Title: Single-stranded DNA in the bone microenvironment promotes prostate cancer bone metastasis via the ITGA6-FAK pathway

doi: 10.1038/s42003-026-09929-9

Figure Lengend Snippet: A Schematic of the ITGA6 pull-down assay and LC-MS/MS analysis. B Mass spectrogram of the FAK protein tandem mass spectrum. C Co-immunoprecipitation (co-IP) analysis of the interaction between FAK and ITGA6 with or without EHBP1-ssDNA treatment performed using an antibody to pull-down extracted proteins from PC-3 cells. Western blotting analysis ( D ) of ITGA6, ITGB4, p-FAK(Try397), p-AKT, p-ERK, FAK, AKT, and ERK protein levels in PC-3 cells treated with different concentrations of EHBP1-ssDNA for 4 h, and quantification from three independent experiments ( E ). GAPDH was used as a loading control. Western blotting analysis ( F ) of ITGA6, ITGB4, p-FAK(Try397), p-AKT, p-ERK, FAK, AKT, and ERK protein levels in PC-3 cells treated with 10 nM EHBP1-ssDNA for different times, and quantification from three independent experiments ( G ). GAPDH was used as a loading control. Western blotting analysis ( H ) of p-FAK(Try397), p-AKT, p-ERK, FAK, AKT, and ERK protein levels in sgControl or sgITGA6-transfected PC-3 cells treated with scrambled DNA or EHBP1-ssDNA or blank control, and quantification from three independent experiments ( I ). GAPDH was used as a loading control. Western blotting analysis ( J ) of p-FAK(Y397), p-AKT, p-ERK, FAK, AKT, and ERK protein levels in PC-3 cells treated with or without EHBP1-ssDNA or Defactinib, and quantification from three independent experiments ( K ). GAPDH was used as a loading control. L Representative images of cell adhesion (top), invasion (middle), and migration (bottom) from scrambled DNA, EHBP1-ssDNA, Defactinib + scrambled DNA, Defactinib + EHBP1-ssDNA groups. Scale bar, 100 μm. M Quantitative analysis of cell adhesion, invasion, and migration from each group. n = 5 independent experiments.* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 by one-way ANOVA with Tukey’s post hoc test. The error bar represent standard error of the mean.

Article Snippet: Defactinib (MedChemExpress, 1073154-85-4) at 25 mg/kg was administered via oral gavage once a day for 4 weeks.

Techniques: Pull Down Assay, Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Control, Transfection, Migration