Journal: Theranostics

Article Title: Proteomes from AMPK-inhibited peripheral blood mononuclear cells suppress the progression of breast cancer and bone metastasis

doi: 10.7150/thno.80294

Figure Lengend Snippet: Suppression of tumorigenic behaviors of breast cancer cells by CM Lym-Dor . CN = control, CW = CW008 (PKA activator), Dor = Dorsomorphin (AMPK inhibitor), AICAR (AMPK activator), PBMC = peripheral blood mononuclear cells, and CM = conditioned medium. The double asterisks indicate p < 0.01. (A) Suppression of MTT-based viability of MDA-MB-231, MDA-MB-436, and MCF7 breast cancer cells by CM Lym-CW and CM Lym-Dor , respectively. (B&C) Suppression of scratch-based migration, and transwell invasion of MDA-MB-231 cells by CM Lym-Dor . (D) Reduction in p-Src, Snail, and PDL1, together with the elevation in c-Cas3 (cleaved caspase 3) in MDA-MB-231 cells by CM Lym-Dor . (E&F) Suppression of MTT-based viability, and transwell invasion of MDA-MB-231 cells by CM PBMC-Dor , which was derived from peripheral blood mononuclear cells with Dorsomorphin.

Article Snippet: Antibodies against p-Src, Src, Snail, cleaved-Caspase 3, Caspase 3, PDL1, ENO1, MSN, CD44, Mtdh, and PABPC1 (Cell Signaling), NFATc1, cathepsin K (Santa Cruz, Dallas, TX, USA) and β-actin as a control (Sigma) were employed.

Techniques: Migration, Derivative Assay

Journal: Theranostics

Article Title: Proteomes from AMPK-inhibited peripheral blood mononuclear cells suppress the progression of breast cancer and bone metastasis

doi: 10.7150/thno.80294

Figure Lengend Snippet: Additive tumor-suppressing effects by Taxol and CM Lym-Dor in a mouse model of the primary tumor. pl = placebo, Dor = Dorsomorphin (AMPK inhibitor), and CM = conditioned medium. The double asterisk indicates p < 0.01. (A) Dose responses of CM Lym-Dor . The green and red arrowheads indicate the concentration of CM Lyn-Dor for the in vitro and in vivo experiments, respectively. (B) Dose responses of Taxol with and without CM Lym-Dor . The doses of 2 nM and 75 nM are IC50 (50% inhibition) for Taxol with and without CM Lyn-Dor . (C) Additive reduction in the size and weight of mammary tumors by Taxol and CM Lym-Dor in NOD/SCID/γ (-/-) (NSG) female mice. (D) Reduction in p-Src and Snail, Mtdh, and the elevation in c-Cas3 (cleaved caspase 3) in the tumor tissues in tumor-inoculated mice that were treated with Taxol or CM Lym-Dor . (E) Increase in body weight by CM Lym-Dor .

Article Snippet: Antibodies against p-Src, Src, Snail, cleaved-Caspase 3, Caspase 3, PDL1, ENO1, MSN, CD44, Mtdh, and PABPC1 (Cell Signaling), NFATc1, cathepsin K (Santa Cruz, Dallas, TX, USA) and β-actin as a control (Sigma) were employed.

Techniques: Concentration Assay, In Vitro, In Vivo, Inhibition

Journal: Theranostics

Article Title: Proteomes from AMPK-inhibited peripheral blood mononuclear cells suppress the progression of breast cancer and bone metastasis

doi: 10.7150/thno.80294

Figure Lengend Snippet: Role of the Enolase 1 (ENO1)/Moesin (MSN)-Metadherin (Mtdh) regulatory axis. CN = control, CM = conditioned medium, NC = negative control, si = siRNA, and Dor = Dorsomorphin. The double asterisk indicates p < 0.01. (A&B) Elevated levels of ENO1 and MSN in CM Lym-Dor . (C&D) Immunoprecipitation of Mtdh by ENO1 and MSN. (E) Reduction in the efficacy of ENO1 and MSN in Mtdh-silenced MDA-MB-231 cells. (F) Reduction of p-Src and Snail by the application of ENO1 and MSN recombinant proteins, and its suppression by silencing Mtdh. (G&H) Reduction in MTT-based cell viability and scratch-based migration of MDA-MB-231 breast cancer cells by Mtdh overexpressing Jurkat cell-derived CM.

Article Snippet: Antibodies against p-Src, Src, Snail, cleaved-Caspase 3, Caspase 3, PDL1, ENO1, MSN, CD44, Mtdh, and PABPC1 (Cell Signaling), NFATc1, cathepsin K (Santa Cruz, Dallas, TX, USA) and β-actin as a control (Sigma) were employed.

Techniques: Negative Control, Immunoprecipitation, Recombinant, Migration, Derivative Assay

Journal: Theranostics

Article Title: Proteomes from AMPK-inhibited peripheral blood mononuclear cells suppress the progression of breast cancer and bone metastasis

doi: 10.7150/thno.80294

Figure Lengend Snippet: Putative regulatory mechanism for the action of CM Lym-Dor . (A) Reduced %survival of all types of cancer patients with high mRNA levels of ENO1, MSN, and Mtdh. (B) Lowered survival rates of all types of cancer patients with high levels of PABPC1. (C) Pairwise gene expression correlation analysis of PABPC1/MTDH in breast cancer and control. (D) Reduction of Mtdh, p-Src, and Snail with an elevation of cleaved caspase 3 (c-Cas3) in MDA-MB-231 cells by 5 µg/mL PABPC1 recombinant protein. (E) The schematic diagram for the regulatory mechanism of tumor-suppressing action of CM Lym-Dor . Human and mouse samples/molecules are shown in blue and brown, respectively.

Article Snippet: Antibodies against p-Src, Src, Snail, cleaved-Caspase 3, Caspase 3, PDL1, ENO1, MSN, CD44, Mtdh, and PABPC1 (Cell Signaling), NFATc1, cathepsin K (Santa Cruz, Dallas, TX, USA) and β-actin as a control (Sigma) were employed.

Techniques: Expressing, Recombinant

Journal: Cancers

Article Title: ImmunoPET Imaging Identifies the Optimal Timepoint for Combination Therapy in Xenograft Models of Triple-Negative Breast Cancer

doi: 10.3390/cancers15051589

Figure Lengend Snippet: Dasatinib can upregulate the expression of gpNMB on MDA-MB-468 and MDA-MB-231 cells in vitro. ( A ) Western blot for gpNMB, ꞵ-actin, and dasatinib-related proteins p-Src, Src; ( B ) Normalized gpNMB expression relative to ꞵ-actin. DAS = dasatinib.

Article Snippet: Nonspecific binding was blocked with 3% bovine serum albumin (BSA) in TBST, and proteins of interest were probed with the following primary antibodies at 4 °C overnight: goat-anti-human gpNMB (0.5 µg/mL, AF2550 R&D systems); mouse anti-Beta-actin (1:5000, MA1-140, ThermoFisher); rabbit anti-Src (1:500, Cell Signaling Technology, Danvers, MA, USA, cat# 2109); and rabbit anti-Phospho-Src Family (Tyr416) (1:1000, Cell Signaling cat# 6943).

Techniques: Expressing, In Vitro, Western Blot

Journal: Cell & Bioscience

Article Title: ANGPTL2 binds MAG to efficiently enhance oligodendrocyte differentiation

doi: 10.1186/s13578-023-00970-3

Figure Lengend Snippet: ANGPTL2-MAG induces Fyn-mediated signaling to enhance the differentiation of oligodendrocytes. A Gene Ontology (GO) analysis of the downregulated differentially expressed genes (DEGs) in the brains of Angptl2 + / + and Angptl2 −/− mice at day 15 as determined by RNA sequencing (n = 3). B Enrichment score plots from GSEA related to the GO signature for myelin sheath and ensheathment of neurons (n = 3). FDR, false discovery rate; NES, normalized enrichment score. C Relative mRNA levels of potential candidates related to myelination markers, transcription factors, metabolic regulators and other genes in the brain tissues of Angptl2 + / + and Angptl2 −/− mice at day 15 as measured by quantitative RT-PCR (n = 3). D Immunoblot analysis of MYRF and ANGPTL2 protein levels in the brain tissues of Angptl2 + / + and Angptl2 −/− mice at day 5, day 15 and day 35. Ratio of MYRF/β-actin was quantified and normalized against Angptl2 + / + , respectively. One representative experiment is shown. E–F Immunoblot analysis of MYRF protein levels in the brain tissues of Mag + / + and Mag −/− mice ( E ) or Mag −/− Angptl2 + / + and Mag −/− Angptl2 −/− mice ( F ) at day 5, day 15 and day 35. Ratio of MYRF/β-actin was quantified and normalized against Angptl2 + / + , respectively. One representative experiment is shown. G – H MAG directly interacted with FYN, as detected by forward ( G ) or reverse ( H ) co-immunoprecipitation assays. CMV-MAG (full-length)-FC and pLVX-FYN-strepII plasmids were used in this experiment. One representative experiment is shown. I RSC96 cells with ectopic expression of MAG (full-length)-FLAG and FYN-StrepII were treated with ANGPTL2 proteins, followed by co-immunoprecipitation analysis to evaluate the changes in tyrosine phosphorylation levels of MAG and FYN using 4G10 and p-SRC (Tyr416) antibodies, respectively. The levels of immunoprecipitated protein were quantified and normalized against the control group, respectively. One representative experiment is shown. J RSC96 cells overexpressing FYN-StrepII or MAG (full-length)-FC were subjected to immunoblot analysis to determine MYRF protein levels. Ratio of MYRF/β-actin was quantified and normalized against negative control (empty vector), respectively. One representative experiment is shown. K Western blot analysis of the protein levels of P-SRC (Tyr416), Fyn and MBP in HCN cells 72 h after induction with IGF1 (100 ng/ml), with/without ANGPTL2-Flag (2 μg/ml) and AZD0530 (2 μM) as indicated. Ratios of P-SRC (Tyr416)/β-actin, Fyn/β-actin, MYRF/β-actin and MBP/β-actin were quantified and normalized against the control treated with IGF1 alone, respectively. One representative experiment is shown. L Schematic diagram of the working model for the role of ANGPTL2-MAG in oligodendrocytes differentiation, myelination and differentiation. (*** p < 0. 001)

Article Snippet: Samples were separated by SDS-PAGE, transferred to nitrocellulose membranes and immunoblotted with primary antibodies as following: anti-MBP (CST, Cat#78896), anti-MAG (Abcam, ab89780), anti-MOG polyclonal antibody (Santa Cruz Biotech, SC-166172), anti-Fyn (Abcam, ab184276), anti-Phospho-Src family (Tyr416) (CST, Cat #2101), anti-MYRF (Abclonal, A16355), anti-ANGPTL2 (R&D, AF1444) and anti-β-Actin-pAb-HRP-DirecT (MBL, PM053-7).

Techniques: RNA Sequencing Assay, Quantitative RT-PCR, Western Blot, Immunoprecipitation, Expressing, Negative Control, Plasmid Preparation

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: Pharmaceutics

Article Title: Licochalcone A Suppresses Renal Cancer Cell Proliferation and Metastasis by Engagement of Sp1-Mediated LC3 Expression

doi: 10.3390/pharmaceutics15020684

Figure Lengend Snippet: LicA inhibits metastasis via the FAK/Src signaling pathway of RCC cells. Different LicA concentrations (0, 6.25, 12.5, and 12.5 µM) were applied to ACHN cells, and the cells were then left to react for 24 h. ( A ) Western blotting was utilized to examine the expression of p-FAK, t-FAK, p-Src, and t-Src in cell lysates. β-actin served as a internal control. The ACHN cells were treated with 12.5 µM LicA in the presence or absence of ( B ) a FAK inhibitor (PF) or ( C ) an Src inhibitor (PP2) for 24 h. The cell lysates were examined using Western blotting to measure the protein levels of p-FAK, t-FAK, and LC3B. ( D , E ) Next, migration and Matrigel-invasion assays were used to assess migration and invasion. Data represent the mean ± SD of at least three separate experiments. ** p < 0.01, compared to un-treated control (0 µM); # p < 0.05, compared with LicA-treated alone.

Article Snippet: Anti-phosphoryl-FAK (p-FAK), total-FAK (t-FAK), phosphoryl-Src (p-Src) and total-Src (t-Src) antibodies were purchased from Cell Signaling (Danvers, MA, USA).

Techniques: Western Blot, Expressing, Migration

Journal: Pharmaceutics

Article Title: Licochalcone A Suppresses Renal Cancer Cell Proliferation and Metastasis by Engagement of Sp1-Mediated LC3 Expression

doi: 10.3390/pharmaceutics15020684

Figure Lengend Snippet: LicA inhibits RCC tumorigenesis and metastasis via the downregulation of the phosphoryl-Src/-FAK pathway. It does so by decreasing the Sp1 transcription activity, resulting in suppressed LC3B expression.

Article Snippet: Anti-phosphoryl-FAK (p-FAK), total-FAK (t-FAK), phosphoryl-Src (p-Src) and total-Src (t-Src) antibodies were purchased from Cell Signaling (Danvers, MA, USA).

Techniques: Activity Assay, Expressing