Journal: The Journal of Experimental Medicine

Article Title: Macrophages promote anti-androgen resistance in prostate cancer bone disease

doi: 10.1084/jem.20221007

Figure Lengend Snippet: Enzalutamide resistance of bone-metastatic PC can be blocked by SRC-specific inhibitor. (A) Src activity estimated using Src score (see Materials and methods) in RNA-seq data of FACS-purified MycCaP-Bo tumor cells from indicated disease stage/treatment (as described in ). (B) Src score in patient bone metastasis and metastases from other organs. (C) Correlation of Src score with macrophage abundance in patient bone metastasis datasets. (D) Correlation of Src score with ECM score in patient bone metastasis datasets. (E) Correlation of Src score with FN1 expression in patient bone metastasis datasets. (F) Correlation of Src score with ITGA5 expression in patient bone metastasis datasets. (G) Immunoblot showing the level of phosphorylated SRC (pSRC) and total SRC (t-SRC) in MycCaP-Bo cells seeded in wells precoated with 1% BSA (BSA), 1 μg/ml FN1 (FN1-1), and 10 μg/ml FN1 (FN1-10) for indicated time. (H) Immunoblotting showing the level of pSRC and t-SRC in modified MycCaP-Bo cells with doxycycline-induced expression of control shRNA or shRNA-targeting Fn1. The cells were treated with doxycycline (500 ng/ml) for 4 d. (I) Immunoblotting showing the level of pSRC and t-SRC in control MycCaP-Bo cells (Ctrl), MycCaP-Bo cells overexpressing ITGA5 clone 1 (#1) and clone 4 (#4) seeded in wells precoated with 1% BSA (BSA) or 1 μg/ml FN1 (FN1) for 6 h before sample harvest. (J) Immunoblotting showing the level of pSRC and t-SRC in in vivo MycCaP-Bo bone metastasis samples with indicated treatments. (K) Representative BLI images of resistant MycCaP-Bo bone metastasis following eCF506 treatment. (L) BLI quantification of resistant MycCaP-Bo bone metastasis following eCF506 treatment ( n = 8∼10). Data are mean ± SEM; *, P < 0.05; **, P < 0.01; ns, not significant. ANOVA was used in A and B, two-tailed unpaired Student’s t test was used for L, and Pearson correlation analysis was used in C–F. Source data are available for this figure: .

Article Snippet: After blocking in Odyssey Blocking Buffer TBS (927-50000; LI-COR Biosciences), membranes were probed with primary Abs against pSRC (D49G4, #6943; Cell Signaling Technology) or SRC (36D10, #2109; Cell Signaling Technology) overnight at 4°C.

Techniques: Activity Assay, RNA Sequencing Assay, Purification, Expressing, Western Blot, Modification, shRNA, In Vivo, Two Tailed Test

Journal: Clinical Epigenetics

Article Title: Phospholipase C delta 1 inhibits WNT/β‐catenin and EGFR-FAK-ERK signaling and is disrupted by promoter CpG methylation in renal cell carcinoma

doi: 10.1186/s13148-023-01448-2

Figure Lengend Snippet: PLCD1 ectopic expression inhibited oncogenic signaling. a Experimentally expressed PLCD1 in 293 T cells was confirmed by Western blot. b Activity of several oncogenic signaling reporters were evaluated using dual-luciferase reporters assay in 293 T cells. b TCF activity and CCND1 , c-Myc and MMP7 promoter reporter activities in PLCD1 -overexpressing A498 (upper) and HH244 (bottom) cells. d Western blot analyses of total β-catenin, p-β-catenin (Ser552), c-Myc, active β-catenin, cyclinD1, MMP7, PLCD1 and GAPDH. e Western blot analyses of EGFR, ERK1/2, Src, FAK and PLCD1 , with GAPDH as internal control. Histograms indicated quantification of the phosphorylated protein normalized to corresponding total protein level. f Schematic diagram of roles and mechanisms of PLCD1 in RCC tumorigenesis

Article Snippet: Antibodies used are: anti-mouse IgG-HRP (DAKO, P0161), anti-rabbit IgG-HRP (DAKO, P0448), GAPDH (Millipore, MAB374), Anti-V5-Tag (Invitrogen, R96025), E-cadherin (CST, 4065), vimentin (Sigma: V6630), Twist (Santa Cruz, sc-15393), MMP7 (Thermo Fisher, MS-813-P0), Total EGFR (CST: 54,359), p-EGFR (CST: 3777), Total FAK (CST: 71,433), p-FAK (Tyr397) (CST: 8556), Total SRC (CST: 2191), p-SRC (CST: 59,548), Total β-catenin (CST: 59,548), active β-catenin (Millipore, 05,665), p-β-catenin (Ser552) (CST: 5651), c-Myc (CST: 18,583), cyclinD1 (CST: 55,506), p-ERK1/2 (CST: 9101), Total ERK1/2 (CST: 4695), Cleaved-Caspase3 (CST: 9661), Cleaved-PARP (CST: 9541), Caspase 3 (CST:9504), PARP (CST: 9532), Bax (CST:2772), Bcl-2 (CST: 2872).

Techniques: Expressing, Western Blot, Activity Assay, Luciferase

Journal: Frontiers in Cell and Developmental Biology

Article Title: The short-chain fatty acid butyrate exerts a specific effect on VE-cadherin phosphorylation and alters the integrity of aortic endothelial cells

doi: 10.3389/fcell.2023.1076250

Figure Lengend Snippet: BUT engages Src kinases for specific tyrosine phosphorylation at VEC. (A) HAOECs were treated for 15 min before and during BUT incubation with PP2 (10 μM) or vehicle (DMSO). Subsequently, phospho-VECY731 levels were analyzed by western blotting. Phospho-signals of n = 4 experiments were quantified by densitometry. (B) Immune fluorescent microscopy of HAOECs treated with the vehicle (DMSO) or PP2 and BUT (5 mM) for 1 h and subsequently stained for VEC and general phospho-tyrosine residues. Scale bar (10 μm). (C) Validation of Src KD by RNAi. HAOECs were transfected with Src-specific siRNA or control-siRNA. Cells were lysed and analyzed for Src expression 12 h later by western blotting. (D) Stimulation of Src KD and control cells with BUT (1 mM) for 1 h and analysis of phosho-VECY731 expression as shown in . Quantification corresponds to n = 6 experiments. (E) Phospho-SrcY416 western blotting of BUT-treated or -untreated cells, transfected with Ctrl or FFAR3 siRNA. Data display densiometric quantification of relative phospho-Src signals normalized to the level in BUT-untreated Ctrl siRNA-transfected cells (dashed red line) of n = 3 experiments.

Article Snippet: We used the following commercially available antibodies for western blotting and immunofluorescence microscopy: monoclonal mouse anti-p-Tyr (PY99, Santa Cruz, sc-7020), polyclonal rabbit anti-phospho-VEC (against phospho-Tyr731, Invitrogen 44-1145G) , polyclonal rabbit anti-phospho-VEC (against phospho-Tyr685, Abcam, ab119785) , polyclonal rabbit anti-phospho-VEC (against phospho-Tyr658, Invitrogen, 44-1144G) , monoclonal mouse anti-human VEC (clone F-8, Santa Cruz, sc-9989), monoclonal mouse anti-human VEC (BV6, Millipore, MABT134), monoclonal mouse anti-human SRC (Invitrogen, AHO1152), monoclonal rabbit anti-human phospho-SRC (against phospho-Tyr 416 (D49G4), Cell Signaling, 6943), monoclonal rabbit anti-human VEC (E6N7A, Cell Signaling, 93467S), and monoclonal mouse anti-human ß-actin (Cell Signaling, 3700).

Techniques: Incubation, Western Blot, Microscopy, Staining, Transfection, Expressing