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: PLOS ONE

Article Title: Combining MEK and SRC inhibitors for treatment of colorectal cancer demonstrate increased efficacy in vitro but not in vivo

doi: 10.1371/journal.pone.0281063

Figure Lengend Snippet: Western blots performed to detect changes in the levels of the signaling factors pSRC, pFAK, and pERK in multiple KRAS-mutated CRC cell lines are shown. Changes in phosphorylation levels were compared with their respective total levels. Vinculin was used as a loading control. C, control; T, trametinib; D, dasatinib; T+D, trametinib and dasatinib. The numbers below the blots denote the phospho-protein levels relative to total protein levels in each sample (Control cells standardized to 1).

Article Snippet: Antibodies against pERK1/2 (cat. #9101; RRID:AB_331646), ERK1/2 (cat. #4695; RRID:AB_390779), pAKT (cat. #9271; RRID:AB_329825), AKT (cat. #9272; RRID:AB_329827), pSRC (cat. #6943; RRID:AB_10013641), SRC (cat. #2123; RRID:AB_2106047), pFAK (cat. #3284; RRID:AB_10831810), FAK (cat. #3285; RRID:AB_2269034), pMEK (cat. #9154; RRID:AB_2138017), MEK (cat. #9126; RRID:AB_331778), PARP (cat. #9542; RRID:AB_2160739), cleaved PARP (cat. #9541; RRID:AB_331426), caspase 3 (cat. #9665; RRID:AB_2069872), and cleaved caspase 3 (cat. #9661; RRID:AB_2341188) were obtained from Cell Signaling Technology.

Techniques: Western Blot

Journal: Cancers

Article Title: Evaluating the RIST Molecular-Targeted Regimen in a Three-Dimensional Neuroblastoma Spheroid Cell Culture Model

doi: 10.3390/cancers15061749

Figure Lengend Snippet: Neoplastic signaling pathway response to R+D ‘pre-treatment’ comparing 2D and 3D cultures. Immunoblot (IB) analysis showing the phosphorylation status of ribosomal protein S6 kinase (p70S6K), translation initiation factor 4E-BP and tyrosine kinase Src under control (C)- and Rapamycin plus Dasatinib (R+D)-treated conditions in two-dimensional (2D) monolayer and three-dimensional (3D) spheroid cultures of neuroblastoma cell line SK-N-AS and SK-N-BE(2). Numbers below bands state phosphoprotein (P) normalized to total protein expression. GAPDH was applied as loading control. C: Vehicle-treated control cells. R+D: Rapamycin plus Dasatinib treatment as depicted in . Star (*): unknown band. MycN-non-amplified (MNN), MycN-amplified (MNA). IB: Immunoblot. Original immunoblot (IB) images .

Article Snippet: The following antibodies were applied: 4E-BP1 (Cell Signaling, #9644), phospho-4E-BP1(Ser65, Cell Signaling, #9456), MycN (Cell Signaling, #9405), p70S6 kinase (Cell Signaling, #9202), phospho-p70S6 kinase (Thr389, Cell Signaling, #9234), Src kinase (Cell Signaling, #2108), phospho-Src (Tyr416, Cell Signaling, #2101), GAPDH (Santa Cruz, #25778), CXCR4 (Abcam, #124824), BMI1 (Cell Signaling, #5856), NANOG (Cell Signaling, #4903), La protein (antibody 3B9 [ ]) and phosho-La protein [ ].

Techniques: Western Blot, Expressing, Amplification

Journal: Nature Communications

Article Title: Fibroblast growth factor 18 alleviates stress-induced pathological cardiac hypertrophy in male mice

doi: 10.1038/s41467-023-36895-1

Figure Lengend Snippet: a The corresponding Src family kinases correlated with the non-hypertrophic myocardium and hypertrophic myocardium were visualized by the heatmap in the normal and ISO treatment samples in GSE18801 dataset. The darker shade of red or blue represents the higher correlation level. b Circle plot depicting the important signal pathways associated with related genes. c NRCMs were treated with ISO (10 μM) in the presence or absence of FGF18 (50 ng/ml) for 48 h. Protein bands at ~60 kDa were excised from the SDS-PAGE gels for LC-MS/MS analysis. d NRCMs were treated with FGF18 (50 ng/ml) for 10 min. The cell lysates were subjected to immunoprecipitation with FGFR3 antibody, followed by immunoblotting with the FYN antibody. Cell lysates were also subjected to immunoprecipitation with IgG as negative control. n = 4. e NRCMs were treatment with FGF18 (50 ng/ml) for 10 min. The cell lysates were subjected to immunoprecipitation with FYN antibody, followed by immunoblotting with the p-SrcY416 and FGFR3 antibodies. Cell lysates were also subjected to immunoprecipitation with IgG as a negative control. n = 4. All numbers ( n ) are biologically independent experiments. Source data are provided as a Source data file.

Article Snippet: The primary antibodies were p-p38 (CST, 4511, 1:1000), p38 (CST, 8690, 1:2000), p-Erk (CST, 9101, 1:2000), Erk (CST, 9102, 1:2000), p-JNK (CST, 4668, 1:1000), JNK (Abcam, ab179461, 1:2000), Bax (CST, 2772, 1:2000), Bcl-2 (Santa cruz, sc-7382, 1:1000), FGF1 (Abcam, ab207321, 1:1000), FGF2 (Santa cruz, sc-74412, 1:1000), FGF3 (Santa cruz, sc-135, 1:1000), FGF5 (Santa cruz, sc-376264, 1:1000), FGF9 (Santa cruz, sc-373716, 1:1000), FGF13 (Affinity biosciences, DF4699, 1:1000), FGF16 (Santa cruz, sc-390547, 1:1000), FGF18 (Santa cruz, sc-393471, 1:1000), FGFR3 (Santa cruz, sc-390423, 1:1000), FYN (Santa cruz, sc-365913, 1:1000), p-Src (CST, 2101, 1:1000), 3-NT (Abcam, ab61392, 1:1000), NOX4 (Origene, TA349083, 1:1000), p22 phox (Santa cruz, sc-271968, 1:1000).

Techniques: SDS Page, Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, Western Blot, Negative Control

Journal: Nature Communications

Article Title: Fibroblast growth factor 18 alleviates stress-induced pathological cardiac hypertrophy in male mice

doi: 10.1038/s41467-023-36895-1

Figure Lengend Snippet: a , b The mice were injected with AAV9-LacZ or AAV9-cTnT-FGF18 intravenously before Sham or TAC operation. a Western blotting with p-FYN (IP: FYN, IB: p-Src) and FYN antibody. Quantification of relative FYN activity and protein expression levels (right panel). n = 5. Data represent means ± SD. one-way ANOVA with Tukey multiple comparisons test. b qRT-PCR analysis of FYN levels. n = 5. Data represent means ± SD. one-way ANOVA with Tukey multiple comparisons test: n.s. = not significant. c – i AAV9-cTnT-FGF18 and control vector were injected intravenously into tail veins of 6 weeks old male C57BL/6J mice, respectively. One week after the injection, these mice were intravenously injected with the adeno-associated virus (AAV9-cTnT-sh-FYN; AAV9-Scramble) into tail veins. One week after the injection, these mice were subjected to Sham or TAC operation for 6 weeks. c Histological analyses of the HE staining and WGA staining ( n = 5. Scale bar = 0.6 mm for upper HE staining; scale bar = 20 μm for lower WGA staining). Statistical results for the sectional cell area (right panel). n = 5. d Real-time quantitative PCR assays. n = 5. e Statistical results for the ratios of HW/BW in the indicated groups. n = 5. f Echocardiographic data for LVEF and LVFS are shown. n = 5. g PSR staining and Quantification (right panel). n = 5. Scale bar = 450 μm, and then zoom in 5 times. h Left ventricular collagen quantification by hydroxyproline assay (μg/mg). n = 5. i Levels of the oxidative damage marker 3-NT. The quantitative analysis of protein immunoblots (right panel), n = 5. j Total ROS levels (by DCFH-DA probe) were quantified. n = 4. k Hydrogen peroxide levels (by Amplex Red assay) quantified in different groups. n = 5. One-way ANOVA was followed by a post hoc Fisher’s comparison test. l Quantification of relative protein levels. n = 5. c – i All quantitative data are reported as means ± SD, one-way ANOVA with Tukey multiple comparisons test. All numbers ( n ) are biologically independent animals. Source data are provided as a Source data file.

Article Snippet: The primary antibodies were p-p38 (CST, 4511, 1:1000), p38 (CST, 8690, 1:2000), p-Erk (CST, 9101, 1:2000), Erk (CST, 9102, 1:2000), p-JNK (CST, 4668, 1:1000), JNK (Abcam, ab179461, 1:2000), Bax (CST, 2772, 1:2000), Bcl-2 (Santa cruz, sc-7382, 1:1000), FGF1 (Abcam, ab207321, 1:1000), FGF2 (Santa cruz, sc-74412, 1:1000), FGF3 (Santa cruz, sc-135, 1:1000), FGF5 (Santa cruz, sc-376264, 1:1000), FGF9 (Santa cruz, sc-373716, 1:1000), FGF13 (Affinity biosciences, DF4699, 1:1000), FGF16 (Santa cruz, sc-390547, 1:1000), FGF18 (Santa cruz, sc-393471, 1:1000), FGFR3 (Santa cruz, sc-390423, 1:1000), FYN (Santa cruz, sc-365913, 1:1000), p-Src (CST, 2101, 1:1000), 3-NT (Abcam, ab61392, 1:1000), NOX4 (Origene, TA349083, 1:1000), p22 phox (Santa cruz, sc-271968, 1:1000).

Techniques: Injection, Western Blot, Activity Assay, Expressing, Quantitative RT-PCR, Plasmid Preparation, Staining, Real-time Polymerase Chain Reaction, Hydroxyproline Assay, Marker, Amplex Red Assay