Fig 1: SR12813 inhibits tyrosine phosphorylation of SFKs proximal to GPVI, CLEC-2 and integrin aIIbß3 receptors. Platelets (4 × 108 cells/ml) were pre-treated with vehicle-control (DMSO 0.1% v/v) or SR12813 (0, 50 and 100 µM) for 20 minutes and stimulated for (A, B) 90 seconds with CRP-XL (1 µg/ml) or (C) 120 seconds with rhodocytin (100 nM) in the presence of indomethacin (20 µM), cangrelor (1 µM), MRS2179 (100 µM) and EGTA (1 mM). (D) Washed platelets (4 × 108 cells/ml), pre-treated with SR12813 (0, 50 and 100 µM) or vehicle-control were exposed to fibrinogen-coated wells (100 µg/ml) of a tissue culture plate and allowed to adhere for 30 minutes. Samples were tested for Src (Y418) or Lyn (Y396) phosphorylation. Representative immunoblots are shown. The phosphorylation levels were quantified and expressed as a percentage of untreated (vehicle) controls. Actin was used as a loading control. Full length blots are shown in supplementary Fig. 9 and 10. Results are mean ± SD (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 was calculated by one-way ANOVA. Figure adapted from corresponding PhD thesis48.
Fig 2: Bletinib inhibits phosphorylation and enzymic activity of SKFs. (a–f) Phosphorylation of SFKs, namely, (a) Src, (b) Lyn, (c) Fgr, and (d) Hck, and downstream proteins, (e) Btk and (f) Vav, was determined through immunoblotting. (g–j) The ADP-Glo kinase assay kit was used to evaluate the enzymic activity. (g) Src, (h) Lyn, (i) Fgr, or (j) Hck (1.5 ng·ml-1) was incubated with DMSO, 1–10 µM bletinib, or 0.1–3 µM PP2, and then 125 µM ATP (substrate) was added to the reaction mixture for 60 min, which was followed by enzymic activity detection. Data are shown as box (interquartile range) and whiskers (min, max) plots, with medians (n = 6). *P < 0.05, significantly different from the DMSO + fMLP group
Fig 3: LYN Is Activated in BRCA1 Null Cells by the Prolyl Isomerase PIN1(A–C) Primary cells from BlgCre Brca1fl/flp53+/- mouse mammary tumors (A), human HCC1937 BRCA1-deficient breast cancer cells (B), and BRCA1 mutant PDX-derived cells (C) were transduced with control (shScr) or Pin1 knockdown (shPin1#1 and shPin1#2) lentiviruses and lysed after 72 hr. Protein extracts were assessed for levels of PIN1, LYN, phospho-LYN (Y397), and c-KIT (Y719) (PDX samples were not probed for phospho-KIT). Representative western blots and quantitation of phospho-LYN (Y397) levels are shown. GAPDH was used as loading control. shScr-, shPin1#1-, and shPin1#2-transduced BlgCre Brca1fl/flp53+/- tumor cells and HCC1937 cells were also seeded at low density in adherent conditions and stained with crystal violet after 6 days. Cell number was determined by absorbance measurement following solubilization of the dye. PDX-derived transduced cells were cultured for 10–12 days in 3D on Matrigel and then assayed for cell viability.(D) Protein extracts from primary BlgCre Brca1fl/flp53+/- mouse tumor cells transduced with vectors carrying wild-type LYNA were subjected to immunoprecipitation by anti-PIN1 or control (IgG) antibodies. Total extracts (input) and immunoprecipitates (IPs) were probed for PIN1 and LYN by western blot.(E) Schematic of LYN showing the position of PIN1 consensus recognition sequences and the proline > isoleucine mutants generated.(F) Representative western blot analysis of LYN phosphorylation levels at the negative regulatory phosphorylation site (Y508) in primary BlgCre Brca1fl/flp53+/- transduced with vectors carrying wild-type LYNA or LYNA proline mutants (LYN P229I, LYN P197I, or LYN P197I P229I).(G) Western blot analysis of LYN autophosphorylation and PIN1 levels in human HCC1937 cells transduced with either control (Ctr) lentivirus or virus-carrying HA-tagged wild-type or mutant BRCA1 (C61G, A1708E, or L1407P).Blots are representative of three independent experiments. Quantitation is shown as mean and SD (n = 3; two-tailed unpaired t tests). *p < 0.05; **p < 0.01; ***p < 0.001. See also Figures S5 and S6.
Fig 4: LYN Is Positively Regulated by c-KIT in Normal Mammary Cells(A) Flow cytometry of primary mammary cells stained with CD45, CD24, and Sca-1 antibodies. CD45+ leukocytes (purple) were gated out (top plot), and CD45- cells (bottom plot) were gated to define basal (CD24+/low Sca-1-, red), luminal ER- (CD24+/high Sca-1-, green), and luminal ER+ (CD24+/high Sca-1+, blue) epithelial cell populations.(B) Expression pattern of c-Kit, Scf, and Lyn splicing transcripts in mouse mammary cell populations. Semiquantitative RT-PCR data are representative of two independent isolates (four mice for each). Amplicons of the expected size using primers spanning the alternative exon for each gene are indicated. Gapdh was used as a control.(C) qRT-PCR gene expression analysis of Scf in mouse mammary cell populations using probes for both total Scf (membrane bound and soluble) or soluble Scf (sSCF) only. Data are from two independent isolates (four mice for each), presented as relative expression levels with leukocytes as the comparators.(D) Schematic of LYN isoforms showing the 21-amino acid insertion (black residues) in the N-terminal domain of LYNA.(E) Representative western blot analysis and quantitation of c-KIT, JAK2, STAT3, AKT, and ERK1/2 phosphorylation levels in protein extracts from primary mouse mammary organoids cultured on Matrigel and stimulated with SCF for the indicated times. Tubulin was used as loading control.(F and G) Representative western blot analysis and quantitation of LYN autophosphorylation (Y397) (F) and immunoprecipitation (IP) LYN kinase assay of protein extracts (G) from primary mouse mammary organoids cultured on Matrigel and stimulated with SCF for 0, 15, 30, and 60 min.(H and I) Western blot of c-Kit expression and LYN autophosphorylation (Y397) in primary mouse mammary organoids after transduction with control (shScr) or c-Kit knockdown (shKit1 and shKit2) lentiviruses (H) or following treatment with c-KIT blocking (ACK2) or immunoglobulin G (IgG) isotype (IgG Ctr) antibodies (I).Unless otherwise stated, blots are representative of three independent experiments (mean and SD; two-tailed unpaired t tests) (in E and F, t tests are relative to time 0). *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S1.
Fig 5: LYN Activity Is Required for Growth of Brca1 Tumor Cells(A) Primary cells isolated from three distinct BlgCre Brca1fl/flp53+/- mouse mammary tumors (1–3) were transduced with control (shScr) or Lyn knockdown (shLyn1 and shLyn2) lentiviruses, seeded at low density in adherent conditions (2D), and stained with crystal violet after 6 days. Viable cell density was determined by absorbance measurement following solubilization of the dye. Representative images of tumor cell colonies at day 6 of culture are shown.(B) shScr-, shLyn1-, or shLyn2-transduced BlgCre Brca1fl/flp53+/- tumor cells (1–3) seeded in Matrigel (3D) were assessed for growth after 6 days. Graphs show cell number assessed at day 5 of culture relative to shScr cells.(C) Ki67 immunofluorescence staining (green) of control (shScr)- and shLyn-transduced BlgCre Brca1fl/flp53+/- tumor cells in 3D culture 6 days after lentiviral transduction. Representative images and quantification of the percentage of Ki67-positive cells (n = 3). Scale bar, 20 µm.(D) Primary BlgCre Brca1fl/flp53+/- mouse mammary tumor cells were transduced with either lentiviral shScr and empty expression vectors (shScr), shLyn and empty expression vectors (shLyn), or shLyn and expression vectors carrying either an shLyn-resistant form (indicated by an asterisk) of wild-type LYNA (shLyn + LYNA*WT) or a kinase-dead LYNA mutant (shLyn + LYNA*KD). LYN protein levels determined by western blot 6 days after transduction. The graph shows cell number assessed at day 5 of culture relative to shScr cells.(E) HCC1937 cells were transduced with control (shScr) or Lyn knockdown (shLyn1 and shLyn2) lentiviruses and tested for LYN expression levels by western blot after 6 days.(F) shScr-, shLyn1-, or shLyn2-transduced HCC1937 cells were seeded at low density in adherent conditions. Viable cell density was determined after 7 days as in (A). Representative images show tumor cell colonies at day 7 of culture.(G) BRCA1 mutant PDX-derived cells (BCM 3887) were transduced with control (shScr) or LYN knockdown (shLyn1 and shLyn2) lentiviruses and tested for cell viability after 10–12 days of culture in 3D on Matrigel.(H) Primary mouse BlgCre Brca1fl/flp53+/- mammary tumor cells were transduced with pHIV-RFP-Tet repressor and pSEW-GFP-TO-H1 (carrying either shScr or shLyn) lentiviruses. Lyn levels were determined in cells transduced with either inducible shScr or shLyn and in either the presence or the absence of doxycycline (DOX) by qRT-PCR relative to shScr cells without DOX.(I) 250,000 inducible shScr- or shLyn-transduced cells were orthotopically injected into the fourth right mammary fat pad of nude mice. These were randomized to DOX treatment or normal diet, and tumor growth was monitored. Tumor volumes were calculated from caliper measurements of tumor width and length. Tumor growth curves (mean ± SEM) and representative images of endpoint tumors are shown.Blots are representative of three independent experiments. Unless otherwise stated, quantitation is shown as mean and SD (n = 3; for PDX cell experiments n = 3 cell isolations from 3 PDX implants in 3 mice; two-tailed unpaired t tests), except for gene expression analysis by quantitative real-time RT-PCR (mean ± 95% confidence intervals; significance of real-time RT-PCR data was determined from confidence intervals; n = 3 independent experiments for each of 3 technical replicates per sample) (Cumming et al., 2007). *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S3.
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